Immunization scheme for variant surface glycoprotein carriers

ABSTRACT

The invention pertains to an immunization scheme for inducing or amplifying an immune response involving Variant Surface Glycoproteins as carriers for antigenic structures against which the immune response is targeted. The invention is based on a specific priming and boosting schedule using the array presented and soluble VSG variants. Surprisingly, the immunization scheme of the invention elicits a strong and long-lasting antibody response in the immunized subject and therefore is applicable in vaccination approaches, immunotherapy and in the production of novel antibodies.

FIELD OF THE INVENTION

The invention pertains to an immunization scheme for inducing or amplifying an immune response involving Variant Surface Glycoproteins as carriers for antigenic structures against which the immune response is targeted. The invention is based on a specific priming and boosting schedule using the array presented and soluble VSG variants. Surprisingly, the immunization scheme of the invention elicits a strong and long-lasting antibody response in the immunized subject and therefore is applicable in vaccination approaches, immunotherapy and in the production of novel antibodies.

DESCRIPTION

Abuse of and addiction to drugs present major problems to medical, social, and economic health, both for individuals and their families, and for society as a whole. Especially in the U.S.A, abuse of prescription opioid pain medications, such as OxyContin® and Vicodin®, has reached epidemic levels and is now a public health crisis of grave concern. In other regions (e.g., Asia, Africa and the Middle East), Tramadol abuse is on a rapid rise (UNODC World Drug Report 2017). Every facet of society has been affected by the relative availability of these drugs, their perceived safety, and the over-prescription of these drugs by medical prescribers.

The path to addiction often derives from medically sanctioned administration for pain management; however, for a substantial fraction of the population (estimated at 10-15%) this can quickly lead to addiction, followed by illicit use of prescription opioids. Of particular concern is the prevalence of illicit use of these drugs by teenagers, and the rapid increase in emergency room visits (reaching 1.2 million in 2009; DAWN report, 2010) as well as in cases of fatal overdoses resulting from abuse (with deaths from overdose, estimated at 72,000 in 2017, surpassing deaths from breast cancer for the first time, with no end in sight). Currently, treatment options are limited to supervised, mediated controlled withdrawal in rehab/detox clinics (for example using methadone or buprenorphine).

Methadone maintenance, developed at the Rockefeller University for the treatment of heroin addiction, remains the most effective and widely used pharmacotherapeutic for any addictive disease (Dole et al., 1966; Kreek, 2000). But treatment often requires years and retention rates are very poor, raising interest in additional options and/or preventatives of abuse in the first place. One approach that has emerged for addiction is a vaccine to raise neutralizing antibodies against a particular drug target (Shen et al., 2012), an option which has been explored most extensively for cocaine (Fox et al, 1996; Shen and Kosten, 2011) and more recently for heroin derivatives (Pravetoni and colleagues, 2012, 2017 and 2018). The idea of immunotherapy to cure opioid use disorder (or at least to prevent overdose) is based on the notion that if a drug conjugated to a carrier protein can elicit a long-lasting immune response, in which antibodies directed to the chemical structure of the drug develop, then circulating antibodies can bind to the drug upon administration by the user, preventing the drug from getting to the site of action in the brain.

Successful vaccination is defined as the elicitation of high antibody titers and memory B cells. High titers are produced by the recruitment of antigen-specific B cell, their activation and to differentiation into antibody-producing plasma blasts. Memory B cells can induce a recall (memory) response which requires, in addition, cognate T-cell help. Most successful vaccines are made of a single component in adjuvant (e.g. a viral particle), that is given as priming immunization and multiple follow-up boosts. But this does not work for vaccines against small molecules or short, well-defined epitopes in isolation. The immune system physiologically responds to large (nano to micrometer) entities (viruses, bacteria) but not to entities the size of a few nucleotides or amino acids. In the context of antibody elicitation against small molecules or defined small epitopes in general, specific B cell recruitment can only be elicited when the small epitope (also known as the hapten) is attached to a much larger protein (known as the carrier), and delivered with adjuvant (which is meant to “artificially” elicit cognate T-cell help). But because haptens are a small fraction of the size of the carrier, most of the immune response is raised against the carrier, and this includes the generation of cognate T-cell help. Consequently, whereas this approach can generate reasonable titers, it has failed in generating memory every time it has been attempted. The use of antigenic particles such as inactivated trypanosomes was also described for VSG-based vaccination using haptens (WO2020/084072).

Thus, it is an objection of the invention to provide a vaccination and immunization approach which allows for the generation of a stable and long-lasting memory immune response in a subject.

BRIEF DESCRIPTION OF THE INVENTION

Generally, and by way of brief description, the main aspects of the present invention can be described as follows:

In a first aspect, the invention pertains to a method for eliciting an immune response, and/or for amplifying a pre-existing immune response, against an antigenic compound in a subject, comprising at least one priming-immunization step and at least one boosting-immunization step, wherein the at least one boosting-immunization step is performed after the at least one priming-immunization step, and wherein: (i) the at least one priming-immunization step comprises an immunization of the subject with an array of immunization-carrier-fusions on a vesicle or bead, and wherein each immunization-carrier-fusion is composed of a carrier protein fused to the antigenic compound; and (ii) the at least one boosting-immunization step comprises an immunization of the subject with the immunization-carrier-fusion used in (i) in a non-arrayed, preferably soluble form.

In a second aspect, the invention pertains to a method for generating antibodies against an antigenic-compound, the method comprising performing in a non-human animal the method of the first aspect, and isolating from the non-human animal newly generated antibodies, or B-cells producing antibodies, wherein the antibodies are directed against the antigenic compound.

In a third aspect, the invention pertains to an antibody, or an antibody producing B-cell, generated and/or isolated according to the method of the second aspect.

In a fourth aspect, the invention pertains to an immunization kit, the immunization kit comprising (a) an array or aggregate of immunization-carrier protein on a vesicle or bead recited in the first aspect and (b) a non-aggregated and/or non-arrayed, preferably soluble form, of the immunization-carrier protein of (a); wherein the immunization-carrier protein of (a) and (b) are capable to be fused to an antigenic compound to obtain an immunization-carrier-fusion recited in the first aspect.

In a fifth aspect, the invention pertains to a method of treating or preventing an adverse condition in a subject by eliciting an immune response, and/or for amplifying a pre-existing immune response, against an antigenic compound which causes or is associated with the adverse condition, the method comprising performing the steps of the method of the first aspect.

DETAILED DESCRIPTION OF THE INVENTION

In the following, the elements of the invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine two or more of the explicitly described embodiments or which combine the one or more of the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

Definitions

The terms “of the [present] invention”, “in accordance with the invention”, “according to the invention” and the like, as used herein are intended to refer to all aspects and embodiments of the invention described and/or claimed herein.

As used herein, the term “comprising” is to be construed as encompassing both “including” and “consisting of”, both meanings being specifically intended, and hence individually disclosed embodiments in accordance with the present invention. Where used herein, “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. In the context of the present invention, the terms “about” and “approximately” denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±20%, ±15%, ±10%, and for example ±5%. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect. Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the”, this includes a plural of that noun unless something else is specifically stated.

The term “antigenic” in context of the invention shall refer to a feature of the particle of the invention to induce a specific immune response when used as an immunogen in a vaccination or immunization procedure.

The term “variant surface glycoprotein”, which is abbreviated as “VSG”, refers to a family of ˜60 kDa proteins which densely pack the cell surface of protozoan parasites belonging to the genus Trypanosoma. The parasite has a large cellular repertoire of antigenically distinct VSGs (˜1500 complete and partial (pseudogenes)) which are expressed from a bloodstream expression site (BES, ES) in a polycistron by RNA polymerase I (recruited to a ribosomal-type promoter) with other ES-associated genes (ESAGs), of which transferrin receptor (Tfr: ESAG6, ESAG7) is one. Only one VSG gene is expressed at a time, as only one of the ˜15 ES are active in a cell. VSG expression is ‘switched’ by homologous recombination induced by double-strand breaks of a silent basic copy gene from an array (directed by homology) into the active telomerically-located expression site. VSG annotation, protein sequences and gene sequences are derivable from public databases such as www.ensemble.org. A collection of VSGs of Trypanosoma brucei brucei (Lister 427 strain) is published in Cross G A. et al., Mol. Biochem. Parasitol. 2014 June; 195(1):59-73. Doi: 10.1016/j.molbiopara.2014.06.004. Preferred VSGs in context of the present invention are T. brucei VSGs, more preferably VSG1, VSG2, VSG3 or ILTat1.24. A VSG according to the invention is preferred which is characterized by an N-terminus which is located 3-dimensionally within the VSG at a position which is, when the VSG is present within a VSG coat, for example on a trypanosome cell, located sufficiently close to the accessible outer coat-surface, that the addition of a linker sequence (preferably not more than 100 amino acids, preferably not more than 50 amino acids, and most preferably not more than 20 amino acids in length) allows for modification of the so extended or not extended N-terminus of the VSG protein. Preferably any sequence that is N-terminal inserted into a VSG sequence will be inserted right before the starting methionine (M). In further embodiments the insertion is immediately downstream of the signal peptide cleavage site. Preferred VSG sequences to be used in context of the invention are the VSGs comprising an amino acid sequence according to any one shown in SEQ ID NO: 1 to SEQ ID NO: 5.

A “VSG type” or “type of VSG” shall pertain to a family member of the VSG family, preferably one of trypanosomal VSGs.

The term “engineered VSG” or “eVSG” shall refer to any VSG protein comprising an artificial modification compared to the wild-type sequence of said VSG. Preferably the eVSG is a non-naturally-occurring sequence. The VSG can be modified to become an eVSG either by post-translational modification, chemical modification, genetic engineering of the VSG coding sequence and any other means known to the skilled person for protein modification.

The terms “immunogenic compound” or alternatively “immunogen” as used herein, encompasses any kind of compound, or structure, capable of eliciting an immune response in a host. Preferably, but not necessarily, an immunogenic compound according to the invention comprises an amino acid sequence or is a small molecular compound. A small molecule in context of the herein described invention shall be any compound having a molecular mass significantly lower than for example complex macromolecules or proteins, so preferably a small molecule has a molecular weight of less than 50 kDa, preferably less than 20 or 10 kDa.

The term “immunization-carrier-fusion” in context of the present invention is a fusion of a carrier protein suitable for immunization purposes, fused to, either covalently, which is preferred, or non-covalently, to an immunogen or antigenic compound for which antibodies or immune responses are to be generated in accordance with the invention. A carrier protein is preferably a VSG or a variant thereof as described herein. The immunogen or antigen is also one as described herein but preferably is a small molecule, such as a small molecule drug.

An “array” or “aggregate” of a carrier protein or immunization-carrier-fusion in accordance with the herein disclosed invention pertains to an ordered arrangement of multiple such proteins in close proximity to each other. The array or aggregate of carrier proteins or immunization-carrier fusions is provided in accordance with the present invention on a vesicle or bead.

Contrary thereto, the term “soluble immunization-carrier-fusion” pertains to solubilized single immunization-carrier-fusion proteins.

The term “sortase,” as used herein, refers to a protein having sortase activity, i.e., an enzyme able to carry out a transpeptidation reaction conjugating the C-terminus of a protein to the N-terminus of a protein via transamidation. The term includes full-length sortase proteins, e.g., full-length naturally-occurring sortase proteins, fragments of such sortase proteins that have sortase activity, modified (e.g., mutated) variants or derivatives of such sortase proteins or fragments thereof, as well as proteins that are not derived from a naturally occurring sortase protein, but exhibit sortase activity. Those of skill in the art will readily be able to determine whether or not a given protein or protein fragment exhibits sortase activity, e.g., by contacting the protein or protein fragment in question with a suitable sortase substrate under conditions allowing transpeptidation and determining whether the respective transpeptidation reaction product is formed.

Suitable sortases will be apparent to those of skill in the art and include, but are not limited to, sortase A, sortase B, sortase C, and sortase D type sortases. Suitable sortases are described, for example, in Dramsi S, Trieu-Cuot P, Bierne H, Sorting sortases: a nomenclature proposal for the various sortases of Gram-positive bacteria. Res. Microbiol. 156(3):289-97, 2005; Comfort D, Clubb R T. A comparative genome analysis identifies distinct sorting pathways in gram-positive bacteria. Infect Immun., 72(5):2710-22, 2004; Chen I, Dorr B M, and Liu D R., A general strategy for the evolution of bond-forming enzymes using yeast display. Proc Natl Acad. Sci. USA. 2011 Jul. 12; 108(28):11399; and Pallen, M. J.; Lam, A. C.; Antonio, M.; Dunbar, K. TRENDS in Microbiology, 2001, 9(3), 97-101; the entire contents of each of which are incorporated herein by reference). Any known sortase can be used as a starting enzyme in an evolution strategy provided herein, and the invention is not limited in this respect. For example, the present invention encompasses embodiments relating to a sortase A from any bacterial species or strain. Those of skill in the art will appreciate that any sortase and any sortase recognition motif can be used in some embodiments of this invention, including, but not limited to, the sortases and sortase recognition motifs described in Ploegh et al., International PCT Patent Application, PCT/US2010/000274, filed Feb. 1, 2010, published as WO 2010/087994 on Aug. 5, 2010; Ploegh et al., International Patent Application PCT/US2011/033303, filed Apr. 20, 2011, published as WO 2011/133704 on Oct. 27, 2011; Liu et al., U.S. provisional Patent Application 61/662,606, filed on Jun. 21, 2012; and Liu et al., U.S. provisional Patent Application 61/880,515, filed on Sep. 20, 2013; the entire contents of each of which are incorporated herein by reference. The invention is not limited in this respect.

The term “sortase substrate,” as used herein refers to a molecule or entity that can be utilized in a sortase-mediated transpeptidation reaction. Typically, a sortase utilizes two substrates—a substrate comprising a C-terminal sortase recognition motif, and a second substrate comprising an N-terminal sortase recognition motif and the transpeptidation reaction results in a conjugation of both substrates via a covalent bond. In context of the invention the “C-terminal sortase recognition motif” is also referred to as “sortagging donor sequence”, whereas the term “N-terminal sortase recognition motif” is referred to as “sortagging acceptor sequence”. In preferred embodiments, the C-terminal and N-terminal recognition motifs are comprised in different amino acid sequences, for example, one N-terminally of the VSG, and the other linked to the immunogen such that there is a free carboxyl group at the end of the sortagging donor site. Some sortase recognition motifs are described herein and additional suitable sortase recognition motifs are well known to those of skill in the art. For example, sortase A of S. aureus recognizes and utilizes a C-terminal LPXT(G/A) motif, preferably LPXTG, (where X is any amino acid and glycine cannot be a free carboxylate) and an N-terminal oligoglycine (G₁₋₅)—the index indicating the number of guanine compounds—, preferably G₃ or G₅, motif in transpeptidation reactions. Additional sortase recognition motifs will be apparent to those of skill in the art, and the invention is not limited in this respect. A sortase substrate may comprise additional moieties or entities apart from the peptidic sortase recognition motif. For example, a sortase substrate may comprise an LPXTG/A motif, the N-terminus of which is conjugated to any agent, (e.g. a peptide or protein, a small molecule, a binding agent, a lipid, a carbohydrate, or a detectable label). Similarly, a sortase substrate may comprise an oligoglycine (G₁₋₅) motif, preferably G₃ or G₅, the C-terminus of which is conjugated to any agent, e.g., a peptide or protein, a small molecule, a binding agent, a lipid, a carbohydrate, or a detectable label. Accordingly, sortase substrates are not limited to proteins or peptides but include any moiety or entity conjugated to a sortase recognition motif.

The terms “sortagging” in context of referring to a donor or acceptor sequence is sometimes also referred to as “sortase donor sequence”, or “sortase acceptor sequence”.

A “pre-ieVSG” in accordance with the invention is an eVSG having a free N-terminal acceptor site for the addition of an immunogenic compound. For example the pre-ieVSG is expressed on the coat of a trypanosome cell and comprises a free sortase acceptor sequence outside (such as accessible on the surface of) the VSG coat. Using a sortase enzyme in a sortase reaction, the pre-ieVSG can be fused to an immunogenic compound which is coupled to a sortase donor sequence.

As used herein the language “pharmaceutically acceptable” excipient, stabiliser or carrier is intended to include any and all solvents, solubilisers, fillers, stabilisers, binders, absorbents, bases, buffering agents, lubricants, controlled release vehicles, diluents, emulsifying agents, humectants, dispersion media, coatings, antibacterial or antifungal agents, isotonic and absorption-delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well-known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary agents can also be incorporated into the compositions.

The term “priming” or “priming immunization” or “priming immunization step” relates to a method step of “priming” immunization, comprising the initial administration of one or more antigens to a subject in preparation for subsequent administration(s) of the same antigen.

Specifically, the term “priming”, as used herein, defines a first immunization using an antigen which induces an immune response to the desired antigen and recalls a higher level of immune response to the desired antigen upon subsequent re-immunization with the same antigen when administered in the context of the same or a different vaccine or immunogen delivery system. Specifically as used in this application, a “priming immunization” refers to the administration of a composition comprising a preparation containing an array of VSG carrier proteins coupled to the antigen, preferably the array is a VSG coat. As used herein, a “priming immunogenic composition or preparation” refers to a preparation containing such VSG carrier antigen fusions of the invention.

Another component of the methods and compositions of the present invention is the use of a “boosting immunization”, or a “boost”, or “boosting-immunization step” which means the administration of a composition delivering the antigen or antigenic compound as encoded in the priming immunization, but utilizing a preparation with a different form of the carrier platform, and preferably is not an array and/or not an aggregate of the VSGs carrier protein antigen fusions described herein, and most preferably is a soluble VSG carrier antigen fusion of the invention, and most preferably of the same VSG; a whole organism or extract and combinations thereof. A boost is sometimes referred to as an anamnestic response, i.e. an immune response in a previously sensitized subject. A boosting immunization or a boosting immunogenic composition can comprise multiple doses, which may be the same or different amounts.

The term “small molecule” or “small molecular compound” or similar expressions is intended to include organic molecules with a molecular weight of 100 Dalton to 10,000 Dalton. Preferably, the small molecule has a molecular weight of 200 to 5,000 Dalton. In particular, the small molecule may be a small chemical fragment. The term “small molecule” is not intended to include fluorescent dyes. Further, the term “small molecule” is not intended to include nucleic acid polymers, large proteins and polysaccharides. However, the building blocks of nucleic acid polymers, large proteins and polysaccharides, i.e. nucleotides, amino acids and monosaccharides, and small oligomers thereof can be considered as small molecules. In particular, the term “small molecule” is intended to include peptides with a size of 250 to 8,000 Dalton.

By “vaccine,” is meant a composition comprising an immunogen or antigenic compound which, when inoculated into a subject, has the effect of stimulating a cellular immune response comprising a T-cell response and/or a humoral immune response comprising a B-cell response generally resulting in antibody production. The T cell response can be a cytotoxic T-cell response directed against an organism that expresses the antigen. However, the induction of a T-cell response comprising other types of T cells by an immunogen disclosed herein is also contemplated. A B-cell response results in the production of antibody that binds to the antigen. The vaccine can to serve to elicit an immune response in the mammal which serves to protect the mammal against a disease. The terms “vaccine,” “immunogen,” “immunogenic polypeptide,”, “antigen”, “antigenic compound” and the like may be used interchangeably. The term “vaccine” does not in any way connote that the composition is capable of fully preventing a disease in a vaccinated subject, or providing any specific or general level of protection against an infectious agent. In certain aspects a “vaccine” can be ineffective in certain subjects while inducing an immune response in other subjects.

In the first aspect, the invention pertains to a method for eliciting an immune response, and/or for amplifying a pre-existing immune response, against an antigenic compound in subject, comprising at least one priming-immunization step and at least one boosting-immunization step, wherein the at least one boosting-immunization step is performed after the at least one priming-immunization step, and wherein: (i) the at least one priming-immunization step comprises an immunization of the subject with an array of immunization-carrier-fusions on a vesicle or bead, and wherein each immunization-carrier-fusion is composed of a carrier protein fused to the antigenic compound; and (ii) the at least one boosting-immunization step comprises an immunization of the subject with the immunization-carrier-fusion used in (i) in a non-aggregated and/or non-arrayed, preferably soluble form.

In the present invention it was surprisingly discovered that immunization procedures involving VSG proteins as carrier proteins for smaller immunogens are highly efficient when during the priming step an array or dense VSG-immunogen fusions are administered, and subsequently, the same VSG-immunogen construct is used in soluble form for one or more boosting steps.

The immunization-carrier-fusion is, preferably, an antigenic particle coated with an engineered variant surface glycoprotein (eVSG), wherein the eVSG comprises the antigenic compound covalently linked, optionally via a linker, to the N-terminus of the VSG carrier protein.

A preferred embodiment of the antigenic particle of the invention includes that the immunogenic compound is covalently linked, optionally via a linker, to the N-terminus of the VSG. The immunogenic compound may be linked to the VSG by any means known to the skilled artisan, including any chemical reaction, preferably click-chemistry, cross-linking, or use of biological entities such as enzymes, ligases, protein-protein interactions and the like. The term “click-chemistry” in context of the invention shall refer to chemistry tailored to generate covalent bonds quickly and reliably by joining small units comprising reactive groups together (see H. C. Kolb, M. G. Finn and K. B. Sharpless, 2001). Any variants of such approaches may be used to connect the immunogenic compound to the VSG in accordance with the invention.

In general, the linking of the VSG to the immunogenic compound may include the use of any linking means or linker, which in context of the herein disclosed invention refers to the means by which the VSG and the immunogenic compound are linked or connected to form an eVSG. The one or more linkers or linking means for linking the VSG and the immunogenic compound may be any structurally suitable means to connect the two. Exemplary linkers include the use of one or more amino acids which may be used to form a peptide, in some embodiments having a modified peptide backbone, a small chemical scaffold, a biotin-streptavidin, an organic or inorganic nanoparticle, a polynucleotide sequence, peptide-nucleic acids, an organic polymer, or an immunoglobulin Fc domain. The means for linking can comprise covalent and/or noncovalent bonds. The one or more linkers can include various sequences or other structural features that provide various functions or properties. For example, the one or more linkers can contain structural elements to allow the eVSG to be derivatized.

In one preferred embodiment of the antigenic particle of the invention, the antigenic particle comprises the linker, which is an N-terminal extension of the wild-type VSG N-terminal sequence, and preferably comprises 5 to 30 amino acids, preferably 10 to 20, more preferably about 15 amino acids. The linker here is used in order to render the N-terminus of the VSG more accessible to modifications, for example using enzymes, when the VSG is provided in context of an assembled VSG coat. The linker sequence is preferably introduced into the VSG by genetic engineering the VSG coding gene. Preferably the linker is introduced immediately C-terminal to, if present, a signal peptide sequence which allows for a cell surface targeting of the VSG protein. Signal peptides are usually cleaved after export. Hence, the term “signal peptide” as used herein refers to amino-terminal amino acid residues that, when attached to a target polypeptide, permits the export of the target polypeptide from the cell and cleavage of the signal peptide. A preferred linker in accordance with the invention is a G4S linker, however, any other protein linker which essentially does not interfere with VSG folding can be used in context of the invention. A G4S linker in accordance with the herein disclosed invention may comprise a multiplicity of consecutive 4GS, such as a preferred linker is a G4S linker which may have the amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 8).

In an additional or alternative embodiment of the invention the VSG may include a sortase acceptor sequence of any sorts. Such a sequence is preferably attached to the N-terminus of the VSG sequence in such a way that it is coat-surface accessible by a sortase enzyme. If the eVSG sequence comprises an N-terminal linker, the sortase acceptor sequence is located at the N-terminus of the linker. Exemplary sortase acceptor sequences are di-Alanine (AA-) or di-Glycine (GG-), or any other sequence that can be used for sortase-mediated ligation.

Hence, in some embodiments of the invention the immunogenic compound is linked to the VSG preferably by using a sortase enzyme.

In particular preferred embodiments of the invention, thus, the antigenic compound forms a linkage to the carrier protein via a sortagging donor sequence, such as (G)3 SLPSTGG and a sortagging acceptor sequence by covalent connection mediated by a sortase. It may be preferred that the linkage composed of the sortagging donor sequence and the sortagging acceptor sequence comprises the sequence -(G)3 SLPSTGAA-, or a sortagging-functional variant thereof.

Hence, in preferred embodiments, the immunization-carrier-fusion has the following (covalent) structure from N- to C-terminus: antigenic compound, a sortagging donor sequence, a sortagging acceptor sequence, a linker, a VSG protein sequence.

The immunization and vaccination protocol of the invention is particularly in application where the antigenic compound is a small molecule drug (of less than 10 kD), such as therapeutic compound, a sugar, an allergen, and/or a dependence causing substance. Even more preferably, the antigenic compound is a dependency causing substance selected from (i) delta-9-tetrahydrocannabinol (THC) or Synthetic cannabinoids, such as classical cannabinoids, non-classical cannabinoids, hybrid cannabinoids, aminoalkylindoles, and eicosanoids; for example 49-THC HU-210, (C8) CP 47,497, JWH-018, AM-2201 (Fluorinated JWH-018), UR-144, XLR-11 (Fluorinated UR-144), APICA, STS-135 (Fluorinated APICA). AB-PINACA, PB-22, 5F-PB-22 (Fluorinated PB-22); or (ii) methamphetamine and derivatives thereof such as 3,4-methylenedioxy-methamphetamine (MDMA)Ecstasy/Molly; or (iii) a synthetic cathinone like alpha-pyrrolidinopentiophenone (alpha-PVP); or (iv) an opioid including heroin, synthetic opioids such as fentanyl or related compounds such as carfentanyl, and other opioid pain relievers, such as oxycodone (OxyContin®), hydrocodone (Vicodin®), codeine, morphine, desomorphine (Krokodil); or (v) steroids (anabolic substances), or is nicotine.

Opium alkaloids and derivatives in accordance with the invention are selected from opium alkaloids: phenanthrenes like codeine; morphine; thebaine; oripavine or mixed opium alkaloids, including papaveretum; esters of morphine like diacetylmorphine (morphine diacetate; heroin); nicomorphine (morphine dinicotinate); dipropanoylmorphine (morphine dipropionate); diacetyldihydromorphine; acetylpropionylmorphine; dmaDesomorphine; methyldesorphine; dibenzoylmorphine; ethers of morphine like dihydrocodeine; ethylmorphine; heterocodeine

Also included are semi-synthetic alkaloid derivatives such as buprenorphine; etorphine; hydrocodone; hydromorphone; oxycodone; oxymorphone.

Also included are synthetic opioids such as anilidopiperidines like fentanyl; alphamethylfentanyl; alfentanil; sufentanil; remifentanil; carfentanyl; ohmefentanyl; also phenylpiperidines like pethidine (meperidine); ketobemidone; MPPP; allylprodine; prodine; PEPAP; promedol.

Diphenylpropylamine derivatives that are included comprise propoxyphene; dextropropoxyphene; dextromoramide; bezitramide; piritramide; methadone; dipipanone; levomethadyl acetate (LAAM); difenoxin; diphenoxylate; loperamide.

Further included are: Benzomorphan derivatives like Dezocine, Pentazocine, Phenazocine; Oripavine derivatives like Buprenorphine, Dihydroetorphine, Etorphine; Morphinan derivatives like Butorphanol; Nalbuphine; Levorphanol; Levomethorphan; Racemethorphan; Others like Lefetamine; Menthol (Kappa-Opioid agonist); Meptazinol; Mitragynine; Tilidine; Tramadol; Tapentadol; Eluxadoline; AP-237; 7-Hydroxymitragynine.

In some embodiments, the immunogenic compound used in context of the invention is an allergen, or any allergy inducing fragment thereof. An “allergen” in context of the herein disclosed invention means any compound capable of eliciting allergy as defined below. An allergen is usually a biopolymer, mostly a peptide or protein, or biological complex sugar molecules, or any fragments or monomers thereof. It is particular preferred to use the present invention for producing antibodies against allergens in host animals. Such antibodies may then be used as a therapeutic against the allergy in a, for example, human subject suffering from the allergy induced by the respective allergen.

The term “allergy” means any type of hypersensitivity reaction to an environmental allergen mediated by immunological mechanisms, including Type I-IV hypersensitivity reactions, including allergic rhinitis, asthma and atopic dermatitis.

In another embodiment the immunogenic compound to be used in context of the invention can be a nucleic acid or a nucleobase derivative or variant, such as variants ogf RNA or DNA nucleobases for which antibodies are needed.

In some additional or alternative embodiments of the invention the antigenic particle comprises a particle which is a vesicle or a bead. Preferably the particle is selected to be capable of being coated by VSG proteins. In some embodiments the trypanosome is an enzyme glycophosphatidylinositol phospholipase C (GPI-PLC)-negative trypanosome. Using a GPI-PLC-negative T. brucei strain has the advantage that after inactivation of the cell, the VSG coat is not disassembled, but remains intact. Thus, in other additional or alternative embodiments the vesicle may be derived from a biological cell that is a non-living, preferably non-infective biological cell, such as an inactivated biological cell, preferably a UV-crosslinked cell.

In some embodiments the antigenic particle of the invention may comprise two or more different eVSGs on its surface. Thereby the antigenic particle may be used to display two or more different (or 3, 4, 5, 6 etc.) immunogenic compounds on its surface coat. For example, the different immunogenic compounds may comprise different immunogenic epitopes of one antigen or may comprise different epitopes of different antigens.

In some other embodiments of the invention the eVSG comprises the immunogen as one or more immunogenic amino acid sequence(s) (that preferably is (are) genetically) inserted into the VSG sequence. Preferably the inserted immunogenic sequence is a xenogenic sequence, and most preferably is inserted into the VSG sequence without causing a deletion in the wild-type VSG sequence. In some embodiments the insertion is located in a surface loop of VSG, such as a region between two secondary structural motifs in the VSG, and preferably wherein said surface loop is located in a 3-dimensional position within the VSG which is surface presented when the VSG is comprised or assembled in a VSG coat, preferably which is surface-accessible and can be presented to the immune system.

In some embodiments of the invention the immunogenic (antigenic) compound comprised in the antigenic particle is a disease-associated antigen, such as a peptide antigen, and the disease is preferably selected from a proliferative disorder, an infectious disease, an inflammatory disorder, an immune deficiency disorder or an autoimmune disorder; or the disease is a non-communicable disease. Most preferably the immunogenic compound is an antigen associated with an infectious disease or proliferative disorder; preferably the antigen is associated with the disease.

A preferred antigenic particle in context of the invention is a living or an inactivated trypanosome cell having an intact VSG coat, wherein the VSG coat comprises one or more immunogenic engineered VSG (ieVSG), preferably a high percentage of an ieVSG (20% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, with increasing preference), wherein the ieVSG comprises an eVSG protein N-terminally linked to an immunogenic compound. Preferably the linkage is provided by a sortase-mediated reaction.

Further provided are nucleic acid constructs comprising an eVSG of the invention suitable for the introduction into the VSG expression locus of trypanosomes. Preferably the nucleic acid construct for introduction into a trypanosome has a structure as shown in any one of FIGS. 3A to 3C. Generally, a nucleic acid of the invention comprises the following elements in this order: a co-transposed region (CTR) of a VSG, for example about 1156 bp preceding the VSG2 open reading frame in Bloodstream Expression Site-1 (BES1) of the T. brucei brucei (Lister 427 strain) genome, immediately followed by the coding region for the eVSG in accordance with the invention, and a telomere seed sequence, for example a telomere seed sequence of about 150 to 250, preferably 200 bp. The construct may further comprise a resistance gene, for example blasticidin, puromycin or hygromycin, preferably following the eVSG coding region. Methods for integrating such constructs into the trypanosome genome are shown in Pinger et al., 2017 (Nature Communications), of which the materials and methods on pages 7 and 8 are incorporated herein by reference.

In other preferred embodiments the antigenic compound is a biological antigen, for example, a peptide or nucleic acid immunogen, wherein the biological antigen is derived from a disease associated antigen structure or molecule. Disease associated antigens may be any antigen that specifically and selectively is expressed in cells or tissue associated with the disease, or in any pathogen causing or being associated with the disease. Such biological antigens may be associated to a cancer or tumour, or may be associated to an autoimmune or inflammatory disease.

Furthermore, biological antigens may be associated with a bacterial, fungal or viral disease, or any other unicellular pathogenic organisms, or a pathogenic or parasitic animal. Typically, such biological antigens are peptide or nucleic acid molecules having a specific sequence only found in the pathogen or disease associated cell or tissue. Preferred peptides are small peptide fragments of a protein expressed as a component of a virus, such as and preferably of a corona virus (such as COVID-19).

Preferred VSG proteins for use in context of the herein disclosed invention are VSG derived from Trypanosoma brucei.

In context of the herein disclosed invention, the array or aggregate of immunization-carrier-fusions constitutes a particle coated with multiple immunization-particle fusions. Hence, such immunization-particle fusions are displayed in an array on the surface of the particle. Preferably an array or aggregate is present if there are more than one immunization-carrier-fusion per particle, preferably more than two, more preferably more than three, or more than 4, 5, 8, 10, 15, 20, more preferably more than 50 such molecules per particle. The density of the immunization-carrier-fusion on the particle will be dependent on the particle used. Higher densities may be obtained by using larger particles, such as an inactivated trypanosome, or a trypanosome coat.

The particle that can be used in context of the present invention is a vesicle or bead. A vesicle as used herein may be a liposome or other micellular or lipid bilayer structure. A bead may be a microspheric bead, such as latex, silica or paramagnetic bead or any other nanoparticle. Beads ranging in size from 0.1 micron to 1 mm can be used in the invention and are therefore encompassed with the term “particle” as used herein. The term particle also encompasses biological cells, as well as beads and other microscopic objects of similar size (e.g., from about 0.1 to 120 microns, and typically from about 1 to 50 microns) or smaller (e.g., from about 0.1 to 150 nm).

In preferred embodiments of the present invention, the immunization-carrier used in the method is a VSG protein, and wherein the VSG protein used as carrier in (i) and (ii) are derived from the same VSG protein type, for example the VSG in both (i) and (ii) are VSG3 of Trypanosoma brucei. The “same” VSG protein in this context shall refer to a mostly identical parent VSG protein, meaning VSG family member, such as the protein sequence used between the carrier used in (i) compared to the carrier used in (ii) shares higher sequences identity to each other as either of them to any given third VSG protein family member. The members of VSG proteins is disclosed for example in Cross G A M, Kim H S, Wickstead B. Capturing the variant surface glycoprotein repertoire (the VSGnome) of Trypanosoma brucei Lister 427. Mol Biochem Parasitol. 2014; 195:59-73 or on “http://tryps.rockefeller.edu”.

In some embodiments that are preferred, the present immunization method comprises at least two priming-immunization steps, and wherein all of the priming immunization steps are performed prior to any of the boosting-immunization steps. In some embodiments there might be three or four priming-immunization steps before any boosting-immunization step is performed.

In particular embodiments it is preferred that at least two boosting-immunization steps are preformed, and that all of the boosting immunization steps are performed after (or subsequent to) any of the priming-immunization steps.

More preferred embodiments of the invention relate to the method wherein at least two priming-immunization steps and at least two boosting-immunization steps are performed. Again, any of the priming-immunization steps are performed prior to any of the boosting-immunization steps. Some preferred embodiments may include any combination of repeated priming and boosting immunization step.

A characteristic of the herein disclosed invention is that the method of the invention elicits a long-lasting, such as more than 1 preferably more than 2 months, antibody response (with antibody titers preferably higher than 100 □g/mL) in the subject that is immunized. Hence, the method is preferably a method for eliciting a long-lasting, such as more than 1 preferably more than 2 months, antibody response (with antibody titers preferably higher than 100□g/mL) in the subject that is immunized.

A subject according to the invention is in particular embodiments selected from an animal having an adaptive immune system, such as vertebrates such as birds (chicken), and mammals such as mouse, rabbit, camel, goat, rat, dog, cat, monkey, hamster or other mammals, or is a human, such as a human patient in need of an elicited or amplified immune response.

In context of the invention the immunogenic compound is a small molecule, a nucleic acid, or a peptide. In other embodiments the immunogenic compound is considered as a small molecule, a nucleic acid, a carbohydrate, a lipid or a peptide, or any combination of these or other chemical entities).

A preferred embodiment of the antigenic particle of the invention includes that the to immunogenic compound is covalently linked, optionally via a linker, to the N-terminus of the VSG. The immunogenic compound may be linked to the VSG by any means known to the skilled artisan, including any chemical reaction, preferably click-chemistry, cross-linking, or use of biological entities such as enzymes, ligases, protein-protein interactions and the like. The term “click-chemistry” in context of the invention shall refer to chemistry tailored to generate covalent bonds quickly and reliably by joining small units comprising reactive groups together (see H. C. Kolb, M. G. Finn and K. B. Sharpless, 2001). Any variants of such approaches may be used to connect the immunogenic compound to the VSG in accordance with the invention.

In general, the linking of the VSG to the immunogenic compound may include the use of any linking means or linker, which in context of the herein disclosed invention refers to the means by which the VSG and the immunogenic compound are linked or connected to form an eVSG. The one or more linkers or linking means for linking the VSG and the immunogenic compound may be any structurally suitable means to connect the two. Exemplary linkers include the use of one or more amino acids which may be used to form a peptide, in some embodiments having a modified peptide backbone, a small chemical scaffold, a biotin-streptavidin, an organic or inorganic nanoparticle, a polynucleotide sequence, peptide-nucleic acids, an organic polymer, or an immunoglobulin Fc domain. The means for linking can comprise covalent and/or noncovalent bonds. The one or more linkers can include various sequences or other structural features that provide various functions or properties. For example, the one or more linkers can contain structural elements to allow the eVSG to be derivatized.

In a second aspect, the invention pertains to a method for generating antibodies against an antigenic-compound, the method comprising performing in a non-human animal the method of the first aspect, and optionally in any single or combination of the described embodiments and variations of the method of the first aspect, and isolating from the subject (such as a non-human animal) newly generated antibodies, or B-cells producing antibodies, wherein the antibodies are directed against the antigenic compound.

In a third aspect, the invention pertains to an antibody, or an antibody producing B-cell, generated and/or isolated according to the method of the second aspect.

The method of the invention in the second and third aspect provides the vaccination procedure described herein for the production of antibodies against a target antigenic compound. It was surprisingly discovered that immunization according to the herein described methods yields stable antibody responses. Hence, the method of the invention is particular useful for generating new antibodies against antigens that otherwise insufficiently immunogenic.

The method of the second aspect preferably comprises a step of producing a hybridoma cell with the isolated B-cell, and/or, a step of isolating paired heavy and light chain antibody genes and recombinantly producing them in mammalian cells.

As used herein, the term “antibody” may be understood in the broadest sense as any immunoglobulin (Ig) that enables binding to its epitope. Full length “antibodies” or “immunoglobulins” are generally heterotetrameric glycoproteins of about 150 kDa, composed of two identical light and two identical heavy chains. Each light chain is linked to a heavy chain by one covalent disulphide bond, while the number of disulphide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulphide bridges. Each heavy chain has an amino terminal variable domain (VH) followed by three carboxy terminal constant domains (CH). Each light chain has a variable N-terminal domain (VL) and a single C-terminal constant domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to cells or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Other forms of antibodies include heavy-chain antibodies, being those which consist only of two heavy chains and lack the two light chains usually found in antibodies. Heavy-chain antibodies include the hcIgG (IgG-like) antibodies of camelids such as dromedaries, camels, llamas and alpacas, and the IgNAR antibodies of cartilaginous fishes (for example sharks). And yet other forms of antibodies include single-domain antibodies (sdAb, called Nanobody by Ablynx, the developer) being an antibody fragment consisting of a single monomeric variable antibody domain. Single-domain antibodies are typically produced from heavy-chain antibodies, but may also be derived from conventional antibodies.

Antibodies (or those from which fragments thereof can be isolated) can include, for instance, chimeric, humanized, (fully) human, or hybrid antibodies with dual or multiple antigen or epitope specificities, antibody fragments and antibody sub-fragments, e.g., Fab, Fab′ or F(ab′)2 fragments, single chain antibodies (scFv) and the like (described below), including hybrid fragments of any immunoglobulin or any natural, synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. VSIR is an immunoglobulin-like protein, and as such it is not (nor its variants) considered—for the purposes of the present invention—an antibody that binds to the antigenic compound of the invention.

Accordingly, in certain embodiments of the invention an antibody produced according to the herein disclosed methods can comprise an antibody heavy chain, or an antigen binding fragment thereof, and/or an antibody light chain, or an antigen binding fragment thereof.

In an alternative, and preferred, embodiment of all ABPs of the invention, the ABP is an antibody or an antigen binding fragment thereof, and the antibody is a monoclonal antibody, or wherein the antigen binding fragment is a fragment of a monoclonal antibody.

Antibodies produced according to the invention are polyclonal or monoclonal antibodies.

The term “monoclonal antibody” or “mAb” as used herein refers to an antibody obtained from a population of substantially identical antibodies based on their amino acid sequence. Monoclonal antibodies are typically highly specific. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (e.g. epitopes) of an antigen, each mAb is typically directed against a single determinant on the antigen. In addition to their specificity, mAbs are advantageous in that they can be synthesized by cell culture (hybridomas, recombinant cells or the like) uncontaminated by other immunoglobulins. The mAbs herein include for example chimeric, humanized or human antibodies or antibody fragments.

Monoclonal antibodies in accordance with the present invention may be prepared by methods well known to those skilled in the art. For example, mice, rats or rabbits may be immunized in accordance with the method of the first aspect. In addition, splenocytes of such immunized animals are harvested as a pool from the animals that are administered several immunisations at certain intervals with test bleeds performed to assess for serum antibody titers. Splenocytes are prepared that are either used immediately in fusion experiments or stored in liquid nitrogen for use in future fusions or cell sorting experiments followed by antibody cloning and expression of humanised monoclonal antibodies (Tiller et al. J. Immunol. Methods 2008, 329(1-2): 112-124). Fusion experiments are then performed according to the procedure of Stewart & Fuller, J. Immunol. Methods 1989, 123:45-53. Supernatants from wells with growing hybrids are screened by e.g. enzyme-linked immunosorbent assay (ELISA) for mAb secretors. ELISA-positive cultures are cloned either by limiting dilutions or fluorescence-activated cell sorting, typically resulting in hybridomas established from single colonies. The ability of an antibody, including an antibody fragment or sub-fragment, to bind to a specific antigen can be determined by binding assays known in the art, for example, using the antigen of interest as the binding partner.

The antibodies described herein may alternatively be prepared through the utilization of the XenoMouse® technology. Such mice are capable of producing human immunoglobulin molecules and antibodies and are deficient in the production of murine immunoglobulin molecules and antibodies. In particular, a preferred embodiment of transgenic production of mice and antibodies is disclosed in U.S. patent application Ser. No. 08/759,620, filed Dec. 3, 1996 and International Patent Application Nos. WO 98/24893, published Jun. 11, 1998 and WO 00/76310, published Dec. 21, 2000. See also Mendez et al., Nature Genetics, 15:146-156 (1997). Through the use of such technology, fully human monoclonal antibodies to a variety of antigens have been produced. Essentially, XenoMouse® lines of mice are immunized according to the VSG technology of the invention, lymphatic cells (such as B-cells) are recovered from the hyper-immunized mice, and the recovered lymphocytes are fused with a myeloid-type cell line to prepare immortal hybridoma cell lines. These hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. Other “humanised” mice are also commercially available: e.g., Medarex—HuMab mouse, Kymab—Kymouse, Regeneron—Velocimmune mouse, Kirin—TC mouse, Trianni—Trianni mouse, OmniAb—OmniMouse, Harbour Antibodies—H2L2 mouse, Merus—MeMo mouse. Also available are “humanised” other species: rats: OmniAb—OmniRat, OMT—UniRat. Chicken: OmniAb—OmniChicken.

The term “humanised antibody” according to the present invention refers to immunoglobulin chains or fragments thereof (such as Fab, Fab′, F(ab′)2, Fv, or other antigen-binding sub-sequences of antibodies), which contain minimal sequence (but typically, still at least a portion) derived from non-human immunoglobulin. For the most part, humanised antibodies are human immunoglobulins (the recipient antibody) in which CDR residues of the recipient antibody are replaced by CDR residues from a non-human species immunoglobulin (the donor antibody) such as a mouse, rat or rabbit having the desired specificity, affinity and capacity. As such, at least a portion of the framework sequence of said antibody or fragment thereof may be a human consensus framework sequence. In some instances, Fv framework residues of the human immunoglobulin need to be replaced by the corresponding non-human residues to increase specificity or affinity. Furthermore, humanised antibodies can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximise antibody performance. In general, the humanised antibody will comprise substantially all of at least one, and typically at least two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanised antibody optimally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, which (e.g. human) immunoglobulin constant region may be modified (e.g. by mutations or glycoengineering) to optimise one or more properties of such region and/or to improve the function of the (e.g. therapeutic) antibody, such as to increase or reduce Fc effector functions or to increase serum half-life. Exemplary such Fc modification (for example, Fc engineering or Fc enhancement) are described elsewhere herein.

The term “chimeric antibody” according to the present invention refers to an antibody whose light and/or heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant regions which are identical to, or homologous to, corresponding sequences of different species, such as mouse and human. Alternatively, variable region genes derive from a particular antibody class or subclass while the remainder of the chain derives from another antibody class or subclass of the same or a different species. It covers also fragments of such antibodies. For example, a typical therapeutic chimeric antibody is a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody, although other mammalian species may be used.

In particular of such embodiments, an antibody produced according to the invention comprises an antigen binding domain of an antibody wherein the antigen binding domain is of a human antibody. Preferably, antibody produced according to the invention comprises an antigen binding domain of an antibody or an antigen binding fragment thereof, which is a human antigen binding domain; (ii) the antibody is a monoclonal antibody, or wherein the antigen binding fragment is a fragment of a monoclonal antibody; and (iii) the antibody is a human antibody or a humanised antibody, or wherein the antigen binding fragment is a fragment of a human antibody, a humanised antibody or a chimeric-human antibody.

Light chains of human antibodies generally are classified as kappa and lambda light chains, and each of these contains one variable region and one constant domain. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon chains, and these define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Human IgG has several subtypes, including, but not limited to, lgG1, lgG2, lgG3, and lgG4. Human IgM subtypes include IgM, and 1gM2. Human IgA subtypes include lgA1 and lgA2. In humans, the IgA and IgD isotypes contain four heavy chains and four light chains; the IgG and IgE isotypes contain two heavy chains and two light chains; and the IgM isotype contains ten or twelve heavy chains and ten or twelve light chains. Antibodies according to the invention may be IgG, IgE, IgD, IgA, or IgM immunoglobulins.

In some embodiments, the antibody of the invention is an IgG antibody or fragment thereof. In some embodiments, the antibody of the invention is an IgE antibody or fragment thereof. In some embodiments, the antibody of the invention is an IgD antibody or fragment thereof. In some embodiments, the antibody of the invention is an IgA antibody or fragment thereof. In some embodiments, the antibody of the invention is an IgM antibody or fragment thereof. Preferably the antibody of the invention is, comprises or is derived from an IgG immunoglobulin or fragment thereof; such as a human, human-derived IgG immunoglobulin, or a rabbit- or rat-derived IgG, and/or an IgG2 immunoglobulin, or fragment thereof. When the antibody of the invention is, comprises or is derived from a rat-derived IgG, then preferably, the antibody is, comprises or is derived from, a rat IgG2a or IgG2b immunoglobulin. When the antibody of the invention is, comprises or is derived from a human-derived IgG, then more preferably, the antibody of the invention is, comprises or is derived from a human IgG1, IgG2 or IgG4, most preferably, the antibody of the invention is, comprises or is derived from a human IgG1 or IgG2

Accordingly, in particular embodiments of the invention, an antibody is preferably an IgG, IgE, IgD, IgA, or IgM immunoglobulin; preferably an IgG immunoglobulin.

An antibody of the invention is preferred, where comprising at least a portion of an immunoglobulin constant region (typically that of a human immunoglobulin) may have such (e.g. human) immunoglobulin constant region modified—for example e.g. by glycoengineering or mutations—to optimise one or more properties of such region and/or to improve the function of the (e.g. therapeutic) antibody, such as to increase or reduce Fc effector functions or to increase serum half-life.

In a fourth aspect, the invention pertains to an immunization kit, the immunization kit comprising (a) an array or aggregate of immunization-carrier protein on a vesicle or bead as recited in the first aspect and (b) a non-aggregated and/or non-arrayed, preferably soluble form, of the immunization-carrier protein of (a); wherein the immunization-carrier protein of (a) and (b) are capable to be fused to an antigenic compound to obtain an immunization-carrier-fusion recited in the first aspect.

In a fifth aspect, the invention pertains to a method of treating or preventing an adverse condition in a subject by eliciting an immune response, and/or for amplifying a pre-existing immune response, against an antigenic compound which causes or is associated with the adverse condition, the method comprising performing the steps of the method of the first aspect.

In context of the present invention, the immunization procedure of the invention is used in context of a vaccination or other medical treatment of an individual. Hence, in that context a further aspect of the present invention then pertains to a pharmaceutical composition comprising an antigenic particle according to the invention together with a pharmaceutically acceptable carrier and/or excipient. A pharmaceutical composition is manufactured for administration to a subject for therapy, prevention or management of a disease or disorder.

A pharmaceutical composition is preferably formulated as a vaccine composition, hence, a composition suitable for vaccination a subject in need of such a treatment.

By way of example, the pharmaceutical composition of the invention may comprise between 0.1% and 100% (w/w) active ingredient, such as about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 8% 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%, preferably between about 1% and about 20%, between about 10% and 50% or between about 40% and 90%. An active ingredient of the composition of the invention is preferably an antigenic particle of the invention.

As used herein the language “pharmaceutically acceptable” excipient, stabiliser or carrier is intended to include any and all solvents, solubilisers, fillers, stabilisers, binders, absorbents, bases, buffering agents, lubricants, controlled release vehicles, diluents, emulsifying agents, humectants, dispersion media, coatings, antibacterial or antifungal agents, isotonic and absorption-delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well-known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary agents can also be incorporated into the compositions.

The pharmaceutical composition of (or for use with) the invention is, typically, formulated to be compatible with its intended route of administration. Examples of routes of administration include oral, parenteral, e.g., intrathecal, intraarterial, intravenous, intradermal, subcutaneous, oral, intraperitoneal, transdermal (topical) and transmucosal administration.

Solutions or suspensions used for parenteral, intradermal, or subcutaneous application, as well as comprising a compound of (or for use with) the invention (e.g. an antigenic particle of the invention), can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine; propylene glycol or other synthetic solvents; anti-bacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Kolliphor® EL (formerly Cre-mophor ELTM; BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the injectable composition should, typically, be sterile and be fluid to the extent that easy syringability exists. It should, typically, be stable under the conditions of manufacture and storage and be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the requited particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminium monostearate and gelatine.

Oral compositions, as well as comprising a compound of (or for use with) the invention (e.g. an antigenic particle of the invention), generally include an inert diluent or an edible carrier. They can be enclosed in gelatine capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystal-line cellulose, gum tragacanth or gelatine; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Stertes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavouring agent such as peppermint, methyl salicylate, or orange flavouring.

Furthermore, the compounds of (or for use with) the invention (e.g. the antigenic particle of the invention) can be administrated rectally. A rectal composition can be any rectally acceptable dosage form including, but not limited to, cream, gel, emulsion, enema, suspension, suppository, and tablet. One preferred dosage form is a suppository having a shape and size designed for introduction into the rectal orifice of the human body. A suppository usually softens, melts, or dissolves at body temperature. Suppository excipients include, but are not limited to, theobroma oil (cocoa butter), glycerinated gelatine, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights, and fatty acid esters of polyethylene glycol.

For administration by inhalation, the compounds of (or for use with) the invention (e.g. an antigenic particle of the invention) are typically delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebuliser.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the pharmaceutical compositions can be formulated into ointments, salves, gels, or creams as generally known in the art.

In certain embodiments, the pharmaceutical composition is formulated for sustained or controlled release of a compound of (or for use with) the invention (e.g. an antigenic particle of the invention). Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

Exemplary unit dosage forms for pharmaceutical compositions comprising the antigenic particles of the invention are tablets, capsules (e.g. as powder, granules, microtablets or micropellets), suspensions or as single-use preloaded syringes. In certain embodiments, kits are provided for producing a single-dose administration unit. The kit can contain both a first container having a dried active ingredient and a second container having an aqueous formulation. Alternatively, the kit can contain single and multi-chambered preloaded syringes.

As mentioned above, preferred pharmaceutical compositions are vaccine compositions. As used herein, the term “vaccine composition” refers to an immunogenic composition which, when administered to a subject, elicits protective immunity against an antigen, in this case the antigen represented by the immunogenic compound. Antibodies produced in accordance with the invention may also be used as therapeutics or passive immunization.

Furthermore, the vaccine composition may include one or more adjuvants. As used herein the term “adjuvant” refers to a compound that, when used in combination with a specific antigenic particle in a formulation, will augment or otherwise alter or modify the resultant immune response. Modification of the immune response can include intensification or broadening the specificity of either or both antibody and cellular immune responses. Modification of the immune response can also mean decreasing or suppressing certain antigen-specific immune responses.

Examples of known adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response, containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminium hydroxide adjuvant. Other known adjuvants include granulocyte macrophage colony-stimulating factor (GM-CSF), Bacillus Calmette-Guérin (BCG), aluminium hydroxide, Muramyl dipeptide (MDP) compounds, such as thur-MDP and nor-MDP, muramyl tripeptide phosphatidylethanolamine (MTP-PE), RIBI's adjuvants (Ribi ImmunoChem Research, Inc., Hamilton Mont.), which contains three components extracted from bacteria, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion. MF-59, Novasomes®, major histocompatibility complex (MHC) antigens are other known adjuvants.

In some embodiments the vaccine composition may include two, three, four, five, six, seven, eight, nine or more antigenic particles, each comprising a difference immunogenic compound derived from one or more antigens.

The herein provided substances, compositions and systems of the invention are preferably for use in the prevention, management, and/or treatment of a medical condition. Hence, the invention relates to a use of the herein disclosed substances, compositions or systems for the manufacture of a medicament of the prevention, management, and/or treatment of a medical condition. Furthermore, the invention relates to a method for prevention, management, and/or treatment of a medical condition, the method comprising the administration of an effective amount of a substances, compositions or system of the invention to a subject in need of such a treatment.

A medical condition which can be prevented, managed or treated in accordance with the invention is in preferred embodiments an addiction to a dependence-causing substance. In this embodiment the immunogenic compound of the antigenic particle is the dependence-causing substance. Without being bound to theory it is expected that the antigenic particles of the invention provide an improved vaccination strategy leading to a strong immune response in a subject and thereby antibodies produced in the subject in response to the antigenic particle will serve as “sponges” to neutralise the dependence-causing substance in the event the subject consumes such substance during a fall-back to the addictive behaviour. Thereby, the vaccination strategy of the invention reduces the positive sensation of the dependence-causing substance and helps the subject to overcome the addiction.

A medical condition which can be prevented, managed or treated in accordance with the invention is in other preferred embodiments an adverse or fatal event caused by drug adulterant. Drug adulterants are substances consumed together with a preparation of a drug of abuse, but often lead to severe overdosing and not-rarely serious adverse effects and even death of the subject. In this embodiment the antigenic particle of the invention includes an immunogenic compound inducing an immune response against a common adulterant in order to protect the abuser from an overdose. Preferably in such a treatment the subject is administered also an antigenic particle of the invention specific for the drug of abuse to which the subject has an addiction.

In further embodiments the invention pertains to a medical condition which is an infectious disease, and wherein the immunogenic compound of the antigenic particle is an immunogenic compound or sequence derived from the infectious organism causing the infectious disease. In preferred aspects the disease is malaria. In other preferred aspects and embodiments, the infectious disease is a viral disease such as a disease caused by a Corona virus, such as Covid-19. With respect to malaria and Covid-19, known and preferred antigens are well known to the skilled person. For example, the immunogen is a compound derived from the viral or plasmodium surface.

Other embodiments of the invention pertain to cancer, and wherein the immunogenic compound of the antigenic particle is a compound associated with or specific for a cancer cell of the cancer.

Other embodiments of the invention pertain to the creation of “slow release depots” (SRDs) for the application of chemotherapeutic agents. SRDs are antibodies or any product of vaccination that bind to specific chemotherapeutic compounds and alter the pharmokinetics or other properties of the treatment for useful purposes (such as reduction of toxicity, prolongation of half-life, alternative tissue tropism, etc.). An example is anti-cancer chemotherapeutic agents (platinum, Adriamycin, etc.) and the generation of antibodies or immune reactions from vaccination that allow for the capture and “slow release” of the chemotherapeutic agents for therapeutic or other useful purposes. Other examples include chemotherapeutics for infectious diseases or allergies for which immunological SRDs provide useful approaches. SRDs for compounds used in industrial applications would also be an embodiment of this invention.

An additional embodiment of the invention pertains to aspects of neurodegenerative diseases and antibodies or any product of vaccination against specific proteins. Examples include: Alzheimer's disease (Amyloid beta (Ab) peptide, Tau), Parkinson's disease (a-Synuclein), Multiple Tauopathies (Tau protein, microtubule-associated), Huntington's disease (Huntingtin with or without tandem glutamine repeats), Amyotrophic lateral sclerosis (Superoxide dismutase 1), Spongiform encephalopathies (Prion proteins), Familial amyloidotic polyneuropathy (Transthyretin, wild-type and mutant forms), small peptides like those associated with migraines), and any possible peptide or small molecule targets that are deemed useful, including but not limited to industrial, agricultural, research or diagnostic purposes.

In general, anything for which an antibody or generalized immune response has shown or could reasonably be expected to potentially show usefulness in a medical, diagnostic or industrial settings qualifies as an embodiment of this invention.

The medical use of the antigenic particle of the invention usually involves the administration of the antigenic particle to a subject in need of the prevention, management, and/or treatment of the medical condition, for example in the form of a vaccine composition as described herein before.

Yet another aspect of the present invention then pertains to a method for the generation of an antibody which is capable of binding to an immunogenic compound, the method comprising the steps of providing an antigenic particle according to the invention, wherein the immunogenic compound of the antigenic particle is the immunogenic compound, or immunogenic parts thereof, the antibody to be generated is capable to bind to; immunizing an antibody-producing human or non-human animal with the antigenic particle; isolating from the immunized human or animal immune cells producing antibodies against said immunogen, and optionally, isolating from said cells said so generated antibodies.

In context of the herein disclosed invention the non-human animal is preferably selected from mouse, rabbit, camel, chicken goat, rat, dog, cat, monkey, hamster or other mammals.

Yet a further aspect then pertains to a method of vaccinating a subject in need of an enhanced immune response specific for an immunogenic compound, the method comprising administering to the subject an amount of an antigenic particle according to the invention which is sufficient to induce an immune response in the subject against the immunogenic compound, wherein the immunogenic compound comprised in the antigenic particle is identical to or is an immunogenic part of, the immunogenic compound for which the enhanced immune response is specific. Preferred is a method for preventing, managing or treating a medical condition, and wherein the immunogenic compound is associated with the medical condition.

Such adverse condition is for example an addiction, and the antigenic compound is an addiction causing substance.

It is to be understood that application of the teachings of the present invention to a specific problem or environment, and the inclusion of variations of the present invention or additional features thereto (such as further aspects and embodiments), will be within the capabilities of one having ordinary skill in the art in light of the teachings contained herein.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

All references, patents, and publications cited herein are hereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCES

The figures show:

FIG. 1 . Map of plasmids used for VSG engineering. Sequences of pHH-VSG3.G4S-Hyg and pHH-ILTat1.24-G4S-Hyg plasmids are provided in SEQ ID NO: 6 and SEQ ID NO: 7 respectively. (A) pHH-VSG3.G4S-Hyg Plasmid Size: 7204 bp. VSG2-CTR (VSG2 Co-transposed Region): 429-878; VSG3.G4S (S317A): 879-2453; VSG2 3′-UTR (VSG2 3′-Untranslated Region): 2454-2533; Actin 5′-UTR (Actin 5′-Untranslated Region): 2727-2834; Hygro (Hygromycin Resistance Gene): 2854-3879; Aldolase 3′-UTR (Aldolase 3′-Untranslated Region): 3885-4033; Telomere Seed Sequences: 4715-4915; Ori (Bacterial Origin of Replication): 5385-5973; AmpR (Ampicillin Resistance Gene, Beta-lactamase): 6144-7004 (Reverse Complement); AmpR Promotor (Ampicillin Resistance Gene Promoter, Beta-Lactamase Promoter): 7005-7109 (Reverse Complement); (B) pHH-ILTat1.24-G4S-Hyg Plasmid Size: 7219 bp. VSG2-CTR (VSG2 Co-transposed Region): 429-878; ILTat1.24-G4S: 879-2468; VSG2 3′-UTR (VSG2 3′-Untranslated Region): 2469-2548; Actin 5′-UTR (Actin 5′-Untranslated Region): 2742-2849; Hygro (Hygromycin Resistance Gene): 2869-3894; Aldolase 3′-UTR (Aldolase 3′-Untranslated Region): 3900-4048; Telomere Seed Sequences: 4730-4930; Ori (Bacterial Origin of Replication): 5400-5988; AmpR (Ampicillin Resistance Gene, Beta-lactamase): 6159-7019 (Reverse Complement); AmpR Promotor (Ampicillin Resistance Gene Promoter, Beta-Lactamase Promoter): 7020-7124 (Reverse Complement). (C) The sequence of pPNSM2, importantly encoding “sortaggable VSG3” (synonymous with “VSG3-G4S (S317A)”) is provided in SEQ ID NO: 6. pPNSM2 Plasmid Size: 7060 bp. Key restriction sites for cloning and cell line generation are marked, with the bold sites representing the two options for linearization prior to transfection. The key elements of the plasmid are also marked, with the following coordinates in order: BES1 Co Transposed Region (1-1227, acting as a homologous region to facilitate genomic integration), Sortaggable VSG3 (1247-2821), VSG 3UTR (2828-2896), Actin 5′-UTR (3087-3193), Hygro (3219-4244, delineating the hygromycin resistance gene open reading frame), Aldolase 3′-UTR (4255-4395), ori (5020-5608, delineating the bacterial origin of replication on the bottom strand), AMP R (5779-6639, delineating the bacterial ampicillin resistance cassette on the bottom strand), and telomere seeds (6785-6985). Plasmids for VSG2 and ILtat1.2 can be generated based on the plasmids described for VSG3 above by replacing the sortaggable VSG3 by VSG2 or ILtat1.2.

FIG. 2 . Crystallographic studies help to determine the accessibility of the VSG N-terminus to the sortase enzyme as illustrated using 5 examples (all published except VSG13). “N” denotes the location of the free N-terminus for each protein. The line denotes a roughly estimated general location of surface-exposed residues (the “top” of the VSG coat, what would be accessible to the sortase enzyme). While many VSGs can be engineered to accept tags through sortase-based conjugation (and the inventors have already done this for VSG2, VSG3 and ILTat1.24 as discussed later), not all can be (for example the N-terminus in VSG13 is far more buried and may not be sufficiently accessible). Structural biology presents a very useful pre-screening of VSGs to uncover many surface elements and other architectural features that inform the choices of VSGs (e.g., the discovery of the O-linked sugar on VSG3 that led the inventors to work with a specific serine to alanine mutant, S317A, to remove it from the surface).

FIG. 3 . Knock-in strategy of sortaggable VSGs into the genome of Trypanosoma brucei. A to C show the genetic cloning strategy to express engineered VSG2, VSG3 and ILTat1.24 from the active expression site of endogenous VSG (replacing wild-type VSG2).

FIG. 4 . Amino acid sequences of the sortaggable VSG proteins. A. Amino acid sequence of the sortaggable VSG3.G4S (S317A) protein. The signal peptide is underlined. The mature, sortaggable VSG3 is derived from a more antigenic mutant of wild-type VSG3 (S317A). VSG3-G4S will initiate with the di-Alanine (in bold and italic). This dipeptide will be the acceptor of the sortase A reaction (and will accept any moiety N-terminally linked to the peptide sequence LPSTGG). The extension of the N-terminus (by addition of the (G₄S)₃ peptide linker, in bold), is crucial for the ability of sortase A to access the di-Alanine. B. Amino acid sequence of the sortaggable VSG2 protein (VSG2-1DK). The signal peptide is underlined. The mature, sortaggable VSG2-1DK will initiate with the di-Alanine (in bold and italic). This dipeptide will be the acceptor of the sortase A reaction (and will accept any moiety N-terminally linked to the peptide sequence LPSTGG). The extension of the N-terminus (by addition of a linker peptide consisting of a TEV protease cleavage site flanked by poly-Glycine, in bold) is crucial for the ability of sortase A to access the di-Alanine. C. Amino acid sequence of the sortaggable ILTat1.24 protein (ILTat1.24-G4S). The signal peptide is underlined. The mature, sortaggable ILTat1.24-G4S will initiate with tetra-Glycine (shown in bold and italic). This tetra-Glycine will be the acceptor of the Sortase A reaction (and will accept any moiety N-terminally linked to the peptide sequence LPSTGG). The extension of the VSG N-terminus (by addition of the (G4S)2 peptide linker, in bold), is crucial for the ability of Sortase A to access the tetra-Glycine.

FIG. 5 . Wild-type T. brucei sheds its VSG coat upon death. A. Cartoon of the process showing that GPI-PLC is activated upon cell death. B. The location of GPI anchor cleavage site is shown. Cleavage of the GPI anchor releases (sheds) VSG from the coat and the coat-less trypanosome disintegrates through osmotic pressure (and consequently losing its immunogenic properties).

FIG. 6 . Illustration of the overall method of sortagging the VSG coat of a Trypanosome. A. Visualization of a VSG protein homodimer embedded in the membrane of a Trypanosome via a glycophosphatidylinositol (GPI) anchor. Bottom: whole trypanosome. Top: Zoom on one VSG protein homodimer. B. Illustration of the sortagging reaction: modified VSG proteins (with an N-terminal di-Alanine), and small molecules (oval) linked to the sortase donor sequence LPSTGG, are covalently linked via a sortase reaction.

FIG. 7 . Methods to detect Sortagging efficiency. A. Top image: Sortagging of VSG2-1DK (left) and VSG3-G4S (S317A) (right) detected via direct fluorescence (6-FAM). Fluorescent microscopic images of a T. brucei cell are shown at top (left: sortagged VSG2-1DK, right: sortagged VSG3-G4S). Bottom image: The 6-FAM sortagged VSGs were also analyzed by flow cytometry analysis using FACSCalibur. B. Sortagging of VGS2-1DK and VSG3-G4S (S317A) detected via FACS analysis of Trypanosomes using a monoclonal antibody against a small-molecule moiety (4-hydroxy-3-nitrophenylacetyl, abbreviated as NP here) followed by staining with an Allophycocyanin (APC)-conjugated mouse monoclonal IgG antibody (B1-8 clone, Abcam). C. A derivatized fentanyl hapten was chemically synthesized and conjugated to the N-terminus of a peptide containing a C-terminal sortase A donor sequence (fentanyl-GGGSLPSTGG, where fentanyl-conjugated compounds are alternatively denoted “Fen-” or “—Fen”). The peptide carrying the fentanyl hapten was conjugated to three different genetically modified VSGs (VSG2, VSG3 and ILTat1.24) using sortase A as described before. Chemical synthesis process of the fentanyl hapten has been described by M. D. Raleigh et al., J. Pharmacol. Exp. Ther. 368:282-291, 2019, and it has been adapted for sortase-mediated conjugation here. D. After sortase A-mediated conjugation of the fentanyl hapten to VSGs, a mouse monoclonal antibody against fentanyl (provided by M. Pravetoni, University of Minnesota) was conjugated to FITC using a kit (Abcam, ab102884) and used to stain the Sortagged VSGs followed by flow cytometry analysis using FACS-Calibur. Non-tagged VSGs were used as control for background staining. Below the graph: The mode and median of the data sets are shown. In both fentanyl and FAM conjugations, ILTat1.24 outperformed VSG2 and VSG3. Also, Sortagging efficiency of VSG3 was moderately higher than VSG2.

FIG. 8 . Comparison of antibody responses to the small molecule 4-hydroxy-3-nitrophenyl acetyl (NP): Immunization with NP-labeled T. brucei vs. the “gold standard” hapten-carrier conjugate (i.e. NP-conjugated chicken gamma-globulin (NP-CGG) in Alum adjuvant). Five 6-8 weeks old female C57BL/6J mice per group were primed at day 0, 3 and 30 with intact VSG3(S317A), or VSG3-NP coats (i.e. U.V-irradiated intact T. brucei cells expressing sortaggable VSG3(S317A), either tagged or not tagged with NP hapten, without adjuvant) or with NP-CGG in Alum adjuvant. These mice received a soluble VSG3(S317A)-NP (in PBS, without adjuvant) booster at day 70 (or were boosted with soluble NP-CGG, in PBS without adjuvant, for the control group). A. The priming immunization with VSG-NP on intact trypanosomes followed by boosting with soluble VSG3(S317A)-NP, shows a clear IgG recall response and results in the generation of substantial IgG titers to the small molecule hapten NP. Titers were measured before and after boost and are shown at serum dilution 1:800. An anti-NP hapten monoclonal IgG (B1-8 clone, Abcam) was serially diluted (4-fold) to cover a range of concentrations from 10 μg/ml to 0.6 ng/ml. Immunization with the conjugated trypanosome coats results in high IgG titers to NP (average ˜500 μg/ml). Furthermore, the fact that soluble VSG3-NP (in PBS, without adjuvant) induced a secondary IgG response strongly indicates that immunization with NP-conjugated VSG 1.0 coats can induce a memory B cell response. While mean titers raised against NP-CGG in Alum are somewhat higher, boosting with soluble NP-CGG (in PBS, without adjuvant) did not induce a robust secondary response, which indicates lack of a memory B cell response after priming with NP-CGG in Alum. B. Immunization using VSG-NP on intact trypanosome coats followed by boosting with soluble VSG3(S317A) NP results in high affinity IgG (as defined by NP2/NP30 IgG titer ratios). Increase in NP2-BSA/NP30-BSA IgG ratio in VSG3-NP group indicates affinity maturation, a hallmark of memory B cell (recall) response. Immunization with the “gold standard” hapten-carrier conjugate (NP-CGG) in Alum provides no increase in affinity of anti-NP IgG antibodies. C. Immunization with VSG-NP on intact trypanosome coats followed by boosting with soluble VSG3(S317A)-NP also yields anti-VSG3 (anti-carrier) antibodies, but those are of a comparable magnitude (in μg/ml) to antisera raised to the NP hapten (as quantified by serial dilutions of an anti-VSG3 mouse monoclonal IgG (11D6 clone). Additional data demonstrate a lack of immunological cross-reactivity between the VSG2 and VSG3 carriers (not shown).

FIG. 9 . Antibody responses to fentanyl elicited by Fen-labeled T. brucei achieve high titers and memory (recall). ELISA measurement of serum IgG against fentanyl hapten and VSG3 carrier protein are shown. A. A tetra-Glycine peptide carrying an N-terminal fentanyl hapten (Fen-G4) was synthesized as described before. The Fen-G4 peptide was conjugated to BSA as a heterologous carrier protein and used to coat 96-well ELISA plates at 10 μg/ml. Five 6-8 weeks old female C57BL/6J mice per group were primed at day 0 and 30 with intact VSG3(S317A), or VSG3(S317A)-Fent coats (i.e. U.V-irradiated intact T. brucei cells expressing sortaggable VSG3(S317A), either tagged or not tagged with Fen-hapten, without adjuvant) via subcutaneous injection. These mice then received a soluble VSG3(S317A)-Fent (in PBS, without adjuvant) booster at day 60. Serum samples tested included the pre-immune (day −4), 2 days before (day 58) and 8 days after (day 68) 1 boost with soluble VSG3(S317A)-fentanyl protein. Anti-fentanyl IgG in sera was detected using an anti-mouse HRP conjugate (1:3000) followed by addition of ABTS substrate and H₂O₂ prepared in citrate-phosphate buffer pH 4.2. Absorption of the samples were measured after 45 min at A405 nm using an ELISA reader (Tecan, Infinite M1000 Pro). B. A mouse monoclonal antibody against Fen-hapten was serially diluted to make a calibration curve in order to quantify IgG concentration in serum samples. Mean±standard deviation of 6 mice per group are shown. C. Similarly, 96-well plates were coated with FPLC-purified VSG3(S317A) protein at 5 μg/well to measure serum IgG against VSG3(S317A) carrier protein. Area under curve (AUC) after 45 min was calculated by GraphPad Prism. The circles, triangles and squares indicate sera at day −4, 58 and 68 respectively. Immunization with the conjugated trypanosome coats results in high IgG titers to fentanyl (average ˜150 μg/ml). Furthermore, the fact that soluble VSG3(S317A)-Fen (in PBS, without adjuvant) induced a secondary IgG response strongly indicates that immunization with Fen-conjugated VSG coats can induce a memory B cell response.

FIG. 10 . Mice immunized with Fentanyl-haptenated T. brucei are protected from intoxication. A. Analgesic activity. Analgesic activity was tested by using the hotplate antinociception assay as described by Cox and Weinstock (1964). Fentanyl effect on hotplate antinociception was tested in unimmunized mice, in mice immunized with carrier only (VSG3(S317A)-only) or in mice immunized with haptenated VSG3(S317A)-Fen. In all cases mice were dosed with a cumulative fentanyl concentration of 0.1 mg/kg (s.c.). Fentanyl was administered subcutaneously every 15-30 minutes at increasing doses and the dose listed is the cumulative dose received. Hotplate antinociception was measured 15 minutes after the final fentanyl dose. Naloxone (0.1 mg/kg, s.c.) was administered 15 minutes after the final fentanyl dose. The effect of fentanyl is shown as latency to response. Fentanyl increased the latency to response after a cumulative dose of 0.1 mg/kg in unimmunized and VSG3-only immunized mice, compared with their baseline values. Naloxone completely reversed fentanyl-induced antinociception in both groups. Mice immunized with VSG3(S317A)-Fen did not show an increase in latency to response, compared to baseline, thus demonstrating that those mice did not get intoxicated by fentanyl at the same dose as the controls. Mean±standard deviation of 5 (unimmunized) or 6 mice per group are shown. B. Straub tail reaction (STR) measured per mouse per group. % denotes number of mice that demonstrated the Straub tail reaction, a dorsiflexion of the tail that is often almost vertical to the orientation of the body or curling back over the animal and stereotyped walking behavior (Bilbey et al, 1960). This phenomenon was first described as a response to opiates in mice (Straub, 1911), and is thought to be mediated by activation of the opioid receptor system because opioid receptor antagonists such as naloxone block the phenomenon (Aceto et al, 1969; Nath et al., 1994; Zarrindast et al, 2001).

FIG. 11 . Each distinct VSG coat elicits a unique subset of B cell specificities (thus, a unique B cell repertoire). Trypanosomes expressing either VSG2 trypanosome coats or the VSG3(S317A) version used in the fentanyl vaccination experiments (a more immunogenic form of VSG3 with serine 317 mutated to alanine) elicit distinct repertoires in C57BL/6 mice. A. Gating strategy for the isolation of plasma cells. Lymphocytes were isolated from spleens and analyzed on a LSR II instrument for plasma cells. Plasma cells were then isolated (single cell sorted) using an ARIA II cell sorter. Cells were stained using an anti-mouse CD19-BV421 and anti-mouse CD138-BV510 antibodies. 7AAD was included in all stainings to exclude dead cells. The data were analyzed using FlowJo v10 software. Ig gene cloning was performed as described before (Tiller et al., 2008). In brief, cDNA of each single cell was generated using random hexamer primers. Ig heavy and corresponding Ig kappa or Ig lambda light chain gene transcripts were amplified using a semi-nested PCR strategy (Tiller et al., 2008). Amplicons were Sanger sequenced and analyzed using NCBI IgBlast. B. V gene repertoires elicited by VSG2 or VSG3(S317A) coated trypanosomes. Histograms summarize the VH and Vκ gene family usage in Ig gene transcripts isolated from plasma cells from C57BL/6 mice exposed to VSG2 or VSG3(S317A) coated trypanosomes (59 and 53 distinct Ig transcripts respectively). Sequences were obtained from 4 and 8 different VH gene families for VSG2 and VSG3(S317A), respectively, and 11 Vκ gene families. Within one bar the different shades of gray show the distribution of different family members within the respective family. The VH10 family, followed by the VH1 family (the largest VH family) was preferably expressed by the majority of plasma cells isolated from the VSG2 mice whereas only very few plasma cells expressing the VH10 family were isolated from the VSG3(S317A) mice. For VSG3 mice, most plasma cells expressed the VH1 family. In the VSG2 mice plasma cells mainly expressed the VK6 family, whereas in VSG3(S317A) mice the Vk family usage was more divers (but mainly VK1, VK4 and VK5). The analyzed sequences clearly demonstrate that the two different VSGs elicit a different VH and Vk family repertoire in plasma cells. This specificity allows one to design optimal vaccination strategies using distinct VSG coat platforms, tailored to the “needs” of specific epitopes. For example, this method can be used to expand an infrequently present B cell that is nevertheless required to be clonally expanded to produce an optimal response against a specific target.

FIG. 12 : A. Illustrates the two types of particles that can be generated from trypanosomes. Immunization with large, intact, inactivated particles generates (FIG. 13 ) a traditional T-independent response. We have discovered that a subsequent boost with soluble VSG particles (i.e. the same material as the initial immunogen but now purified away from the coats in soluble form) is uniquely capable of generating a recall (memory) response together with very high antigen specific IgG titers. B. Shows vesicles derived from trypanosomes exhibiting a VSG coat.

FIG. 13 : shows immunization with haptenated trypanosomes generates high titers of hapten-specific antibodies, and memory. A. Mice primed twice with the NP-haptenated entire VSG coat of trypanosomes followed by a boost with the same moiety (NP-haptenated entire VSG coat of trypanosomes) show a predominantly serum IgM response against NP and no IgG response. B. Mice primed twice with the NP-haptenated entire VSG coat of trypanosomes followed by a boost with the same moiety but now in soluble form show high serum IgG antibody titers again NP and memory. IgG titers are equivalent in proportion against both the carrier as well as the hapten. C. IgG titers elicited by our prime-boost combination are long lasting (e.g. still increasing even one month after the first boost, from Day 68 to Day 98). Data is shown for two different trypanosome strains expressing either VSG2 or VSG3. Data is presented as area under curve (AUC) from a serum dilution range from 1:100 to 1:12800. Each dot represents one mouse (n=5 mice per immunogen).

FIG. 14 : Mice were primed and boosted with either 3E6, 1E7 or 5E7 NP-haptenated VSG3 coats subcutaneously. Mice receiving the highest dose of 5E7 NP-haptenated VSG3 coats showed the lowest NP specific serum IgG titers after the last bleed on Day 100. Thus dose escalation (increase) is not necessarily beneficial and potentially even detrimental to antigen-specific serum IgG titers.

FIG. 15 : The specific prime-boost combination elicits high serum antibody titers against the hapten (here quantified for fentanyl haptenated to VSG3 which represent about 15% of total IgG in a mouse (2.38 mg/ml total IgG in a mouse) mouse (Klein-Schneegans et al., 1989)).

FIG. 16 : Immunization with fentanyl-haptenated trypanosomes generates high titers of anti-Fen antibodies, and memory. A. Experimental timeline. B. ELISA titers presented as “area under curve” (AUC) for a dilution range from 1:100 to 1:12800. The ELISA plate is coated with Fentanyl-BSA (Fen-BSA). The immunogen (Fen-VSG3, VSG3 carrier alone or no immunogen) is noted on the x-axis). Each dot represents antiserum from 1 mouse (n=6 mice per immunogen) (4 time points per immunogen starting with pre-bleed—collected at day −4—up to post-2nd boost—collected at day 98—as per the timeline in A. C. ELISA titers presented as AUC for a dilution range from 1:100 to 1:12800 of antisera against the carrier. The ELISA plate is coated with VSG3. The immunogen (Fen-VSG3, VSG3 carrier alone or no immunogen) is noted on the x-axis.

FIG. 17 : A comparison of vesicles to UV treated coats. Anti-fentanyl IgG normalized AUC is shown over a time course of 50 days. Prime boost on the left side was carried out with UV treated trypanosomal coats whereas vesicles were used on the right side. An apparently stronger increase in IgG levels was found for vesicles. However vesicles are more amenable to GMP production and have a longer shelf life.

The sequences show:

>Tb427VSG-1: GB Accession X56761.2 | Trypanosoma brucei brucei | Lister427 | variant surface glycoprotein MITat 1.1 (Lister 427-1|Not fully assembled by me)| Source = GenBank download 170507| Protein length = 492 SEQ ID NO: 1 MATGRAKNTKWARWLSTAGLIIVVTLPATTMAAER TGLKATAWKPLCKLTTELSKVSGEMLNEGQEVISN IQKIKAAEYKVSIYLAKNPETQALQQLTLLRGYFA RKTNGGLESYKTMGLATQIRSARAAAYLKGSIDEF LNLLESLKGGSENKCLVTTNADTAATRRETKLDDQ ECALSMPETKPEAATRTELTQTGYPNLQHGGGGTA NTFQPTTSTGTCKLLSGHSTNGYPTTSALDTTAKV LAGYMTIPNTQVEATLANMQAMGNGHKATAPAWHE AWEARNREAKAKDLAYTNETGNLDTQPTLKALVKT LLLPKDNTEHNAEATKLEALFGGLAADKTKTYLDM VDAEIIPAGIAGRTTEAPLGKIHDTVELGDILSNY EMIAAQNVVTLKKNLDAVSKKQQTESAENKEKICN AAKDNQKACENLKEKGCVFNTESNKCELKKDVKEK LEKESKETEGKDEKANTTGSNSFLIHKAPLLLAFL LF >Tb427VSG-2: GB Accession X56762.1 | Trypanosoma brucei brucei | Lister427 | variant surface glycoprotein MITat 1.2 (Lister 427-2|Identical in my assembly) | Source = GenBank download 170507 | Protein length = 476 SEQ ID NO: 2 MPSNQEARLFLAVLVLAQVLPILVDSAAEKGFKQA FWQPLCQVSEELDDQPKGALFTLQAAASKIQKMRD AALRASIYAEINHGTNRAKAAVIVANHYAMKADSG LEALKQTLSSQEVTATATASYLKGRIDEYLNLLLQ TKESGTSGCMMDTSGTNTVTKAGGTIGGVPCKLQL SPIQPKRPAATYLGKAGYVGLTRQADAANNFHDND AECRLASGHNTNGLGKSGQLSAAVTMAAGYVTVAN SQTAVTVQALDALQEASGAAHQPWIDAWKAKKALT GAETAEFRNETAGIAGKTGVTKLVEEALLKKKDSE ASEIQTELKKYFSGHENEQWTAIEKLISEQPVAQN LVGDNQPTKLGELEGNAKLTTILAYYRMETAGKFE VLTQKHKPAESQQQAAETEGSCNKKDQNECKSPCK WHNDAENKKCTLDKEEAKKVADETAKDGKTGNTNT TGSSNSFVISKTPLWLAVLLF >Tb427VSG-3: GB Accession AY935575.1 | Trypanosoma brucei brucei | Lister427 | variant surface glycoprotein MITat 1.3 (Lister 427-3|Identical in my assembly) | Source = GenBank download 170507 | Protein length = 509 SEQ ID NO: 3 MQAAALLLLVLRAITSIEAAADDVNPDDNKEDFAV LCALAALANLQTTVPSIDTSGLAAYDNLQQLNLSL SSKEWKSLFNKAADSNGSPKQPPEGFQSDPTWRKQ WPIWVTAAAALKAENKEAAVLARAGLTNAPEELRN RARLALIPLLAQAEQIRDRLSEIQKQNEDTTPTAI AKALNKAVYGQDKETGAVYNSADCFSGNVADSTQN SCKAGNQASKATTVAATIVCVCHKKNGGNDAANAC GRLINHQSDAGANLATASSDFGDIIATCAARPPKP LTAAYLDSALAAVSARIRFKNGNGYLGKFKATGCT GSASEGLCVEYTALTAATMQNFYKIPWVKEISNVA EALKRTEKDAAESTLLSTWLKASENQGNSVAQKLI KVGDSKAVPPAQRQTQNKPGSNCNKNLKKSECKDS DGCKWNRTEETEGDFCKPKETGTENPAAGTGEGAA GANTETKKCSDKKTEGDCKDGCKWDGKECKDSSIL ATKKFALTVVSAAFVALLF >Tb427VSG-13: GB Accession AY935576.1 | Trypanosoma brucei brucei | Lister427 | variant surface glycoprotein MITat 1.13 (Lister 427-13| Not fully assembled by me) | Source = GenBank download 170507 | Protein length = 499 SEQ ID NO: 4 MQRLGTAVFFLLAFRYSTEQAVGLKEPNAPCTTAC GCKSRLLKRLDLYTSKYADGINNERENSEAYSKLV TAALAAVPTMQRKILPLLGAAADILDICRRELATA RPLVQAAISKIEEAAGVYNTLHKLERGLGEAKIEF GGTDLRLTKTKFRATSLGTIHTADCPNADPGETNV KIGLEHEENEPEPAKLITHGHLDATCASGVGQSSS CHTTAVEANTHLTLGLTFSGSSKDESATWNAATNN KRAIHSNDADFLGSNATVAHEALKAIRSAGASTPC SSLITDFNAVRANPKFKLMVIKALLNKPTAEKESD APADEVNNAINSAYGREGSEYNTKTWKDIGSTRIP KADPPGEKTDTIDKLSSLPQWGDAIARLLLQEITK QEEQSIKTSSDEATNKECDKHTAKTEGECTKLGCD YDAENKKCKPKSEKETTAAGKKDRAAGETGCAKHG TDKDKCENDKSCKWENNACKDSSILATKKFALSMV SAAFVTLLF >X56767.1 | Trypanosoma brucei brucei | ILTat1 | mRNA variant surface protein ILTat 1.24 | Source = GenBank download 170421 | Protein length=514 SEQ ID NO: 5 MVYRNILQLSVLKVLLIVLIVEATHFGVKYELW QPECELTAELRKTAGVAKMKVN SDLNSFKTLELTKMKLLTFAAKFPESKEALTLRAL EAALNTDLRALRDNIANGIDRAVRATAYASEAAGA LFSGIQTLHDATDGTTYCLSASGQGSNGNAAMASQ GCKPLALPELLTEDSYNTDVISDKGFPKISPLTNA QGQGKSGECGLFQAASGAQATNTGVQFSGGSRINL GLGAIVASAAQQPTRPDLSDFSGTARNQADTLYGK AHASITELLQLAQGPKPGQTEVETMKLLAQKTAAL DSIKFQLAASTGKKTSDYKEDENLKTEYFGKTESN IEALWNKVKEEKVKGADPEDPSKESKISDLNTEEQ LQRVLDYYAVATMLKLAKQAEDIAKLETEIADQRG KSPEAECNKITEEPKCSEEKICSWHKEVKAGEKNC QFNSTKASKSGVPVTQTQTAGADTTAEKCKGKGEK DCKSPDCKWEGGTCKDSSILANKQFALSVASAAFV ALLF

EXAMPLES

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples.

The examples show:

Example 1: Drug-Decorated VSG Coats

The inventors have generated tools to derivatize the dense and homogeneous surface coat of the African trypanosome (T. brucei) for use as a display platform for (any) antigens to which antibodies need to be raised. These tools consist of:

(a) A specific vector to efficiently replace the expressed VSG (VSG2) with any other VSG of interest (see below). FIG. 1 contains the maps of two such plasmid vectors.

(b) Specific sets of VSGs of interest: these contain extended N-termini that are accessible to the enzyme sortase (here, derived from Streptococcus pyogenes). N-termini accessibility is determined by (i) relative placement on the VSG (determined structurally—FIG. 2 contains VSG2 and VSG3 whose N-termini are accessible, and as a comparison also VSG13, whose N-terminus is not accessible because of steric hindrance). It is also determined by (ii) the initiating amino acids, which must be Ala-Ala for the particular sortase employed (or Gly-Gly for sortases from other organisms).

(c) VSGs that fulfil those criteria are engineered into a modified Lister 427 strain of T. brucei by replacing the active VSG (see (a) above—FIG. 4 contains the amino acid sequences of three such VSGs: VSG3 (A), VSG2 (B) and ILTat1.24 (C); It is worth mentioning that the VSG3 that is engineered for sortagging purposes, contains a mutation (S317A) that removes a native glycosylation event which the inventors have recently shown to be immune-suppressive—Pinger et al., Nat. Microbiology, 2018).

(d) The modification of the Lister 427 strain of T. brucei consists of genetic deletion of the endogenous glycophosphatidylinositol phospholipase C (GPI-PLC), the enzyme that “sheds” VSG off the surface of dying cells (this is crucial to generating T. brucei that can be used as vaccine display platform, because unless GPI-PLC is removed from the genome, any form of inactivation of the parasite (e.g. UV-irradiation), that is crucial (i) to disallow switching and loss of the engineered VSG and (ii) to remove infectivity, will also lead to the disintegration of the VSG coat and of the cell itself (once VSGs are shed due to the action of GPI-PLC, the VSG coat disintegrates and the cells lyse—FIG. 5 explains that concept).

The herein disclosed method depends on “highjacking” the natural ability of T. brucei to elicit a neutralizing (and long-lasting) antibody response to its VSG coat, to produce antibodies at will. The inventors do this by decorating the T. brucei VSG coat not only with any peptide epitope/antigen but also sugars, lipids or small molecules and then using the decorated VSG coat as a vaccine carrier. Specifically, the inventors use the enzyme sortase A to covalently ligate any moiety to VSG coats genetically engineered to carry N-terminal sortase acceptor sequences (FIGS. 4 and 6 ).

Therefore, the inventors produce a His-tagged sortase A, derived from Streptococcus pyogenes, in E. coli, using a plasmid containing the S. pyogenes-derived sortase A expression construct (pSpSortA-pET28a). This plasmid is transformed into BL21 DE3 cells (Life Technologies C6000-03). Colonies from this transformation are used to inoculate large cultures of LB media (Sigma-Aldrich, L3022-1KG) which are then grown shaking at 37° C. to an optical density (OD600) of 0.4-0.8. Cultures are induced with 1 mM IPTG, grown for an additional 3-4 h and harvested by centrifugation. Cell pellets are resuspended in TBS/imidazole (20 mM Tris, 150 mM NaCl, 20 mM imidazole), and lysed using an EmulsiFlex-05 homogenizer (Avestin). DNase-A powder (Sigma D5025) and 5 mM 2-Mercaptoethanol (2-ME) are added to the lysate, which is then clarified by centrifugation to remove particulates. The supernatant is passed through a column packed with Ni-NTA agarose beads (QIAGEN, 30230) equilibrated with Wash Buffer (20 mM Tris, 300 mM NaCl, 20 mM imidazole, 5 mM 2-ME). The column is then washed with 100 ml of Wash Buffer and eluted with 30-35 ml of Elution Buffer (20 mM Tris, 300 mM NaCl, 200 mM imidazole, 5 mM 2-ME). Samples containing protein are then pooled and dialyzed in Dialysis Buffer (20 mM Tris, 150 mM NaCl, 1 mM DTT). The resulting sample is concentrated using a centrifugal filter unit (Amicon Ultra-15, 10,000 NMWL, Merck Millipore), aliquoted and stored at −80° C. for future use.

The sortagging reaction is performed as follows: a mixture of sortagging solution containing 100 uM purified sortase A and 300-600 uM sortaggable-peptide in HMI-9 media is incubated on ice for 30-60 min (a sortaggable peptide includes any peptide with a C-terminal sortase donor sequence, LPSTGG, that can be attached at its N-terminus to another moiety; that moiety can be a fluorophore like 6-FAM, a small molecule like 4-Hydroxy-3-nitrophenyl acetyl (NP) hapten or other small molecules that are drugs of abuse (e.g. fentanyl etc.). GPI-PLC-negative T. brucei cells expressing engineered VSGs are then pelleted, resuspended in the sortagging mixture and incubated for 60 min at 4° C. on an inversion rotator. Cells are then pelleted, washed once with HMI-9 media and pelleted again before final resuspension in HMI-9 media (Hirumi and Hirumi, J. Parasitology, 1989). The efficiency of sortagging can be determined by direct FACS analysis or fluorescence microscopy (e.g. for fluorophores like 6-FAM) or by using specific monoclonal antibodies that bind the moieties decorating the VSG (FIG. 7 contains examples for 6-FAM, NP hapten and fentanyl hapten).

In proof of principle experiments this approach was used to generate (a) robust (in comparison to NP-CGG in Alum adjuvant) and (b) of consistent quality antibodies against a small-molecule hapten (4-hydroxy-3-nitrophenylacetyl or NP) (FIG. 8 ).

This approach can be used for a range of other small molecules (e.g. drugs of abuse like cocaine, nicotine, fentanyl, carfentanyl, tramadol, ketamine etc., but also chemotherapeutics like platinum, Adriamycin etc.; and also small molecules that are industrial by-products of chemical reactions), for toxins that mediate allergic reactions (e.g. aflatoxin and others) for specific peptides that function as important epitopes for infectious diseases (e.g. Plasmodium-derived peptides), for glycosylated or lipidated peptides (e.g. the aberrantly-glycosylated mucin peptides that have been considered as targets for anti-cancer vaccines etc.).

From the perspective of an anti-fentanyl (anti-overdose) vaccine, the major focus is to use this system to vaccinate “at risk” individuals (defined as individuals who are regular users or substance abusers but are not yet addicted/chemically dependent, or addicted individuals leaving rehabilitation centers, as proactive protection against overdose in case of recidivism, which typically will occur within the first two months after leaving rehab). Proof of concept that this has been achieved using the approach herein, is provided in FIGS. 9 and 10 . Additionally, when this approach is coupled to repertoire analysis (FIG. 11 ), it will yield a wealth of anti-fentanyl monoclonal antibodies of varying affinities (directly accessible and ready to reconstitute from the paired immunoglobulin heavy and light chain sequences generated as a result of repertoire sequencing—FIG. 11 ). Such monoclonal antibody “sponges” can be used directly for therapeutic applications (e.g. anti-fentanyl antibody infusion together with methadone maintenance to curb bioavailability as well as cravings and accelerate therapeutic outcomes; or injection in the ER to blunt the effects of overdose in conjunction with naloxone—which acts quickly by antagonizing fentanyl binding to opioid receptors but which is metabolized faster than fentanyl, allowing delayed intoxication).

The Methodology: The ability of trypanosomes to stimulate a robust immune response in the infected individual (a response that is both long-lasting and neutralizing) is well documented. This invention renders this possible, at least in part, due to the discovery that a trypanosome's VSG protein is tolerant to the display of exogenous moieties with high efficiency on its surface using a bacterial transpeptidase sortase-based system (henceforth “sortagging”).

Specifically, a sortase acceptor sequence specific to the sortase (for sortase A derived from Streptococcus pyogenes that is Ala-Ala and for Sortase A derived from Staphylococcus aureus that is Gly-Gly) can be added at the exposed N-terminal part of the VSG protein which, when it gets transported to the surface of the trypanosome, remains accessible to the sortase (see FIGS. 2 and 6 ). It is noted that VSGs initiate with the Methionine of a signal peptide, but that peptide is cleaved upon maturation—hence the mature VSG sequence is not initiating with Methionine. For instance, both VSG2 and VSG3 (the preferred VSG variants) initiate with Ala-Ala, however the exact initiating amino acid must be empirically determined (using Edman degradation, which the inventors have done for both VSGs). Finally, while the endogenous Ala-Ala is present, it is inaccessible to sortase (and requires a short N-terminal extension as shown in FIG. 4 ). A complementary sortase donor sequence is then added C-terminally to the peptide/small molecule of interest (the actual sequence also depends on the sortase used; for sortase A derived from Streptococcus pyogenes, it is LPXTGG). LPXTGG can be added to a small molecule or other moiety with a reactive group (here the inventors use 6-FAM, or the hapten 4-hydroxy-3-nitrophenylacetyl abbreviated as NP, or Fen- FIG. 7 ).

For fentanyl, the N-terminus of the sortase sequence is linked via an amide bond to the fentanyl hapten at the position located in FIG. 7D. While most moieties can be derivatized, a derivatization that retains the antigenicity of the small molecule-peptide conjugate (e.g. NP-GGGSLPSTGG) must be empirically determined (for example this was done through trial and error for nicotine and other small molecules, e.g. NicVax). The sortase then ligates NP-carrying peptide to the exposed N-terminus of the VSG on the surface of the trypanosomes (the inventors validate this using anti-small molecule antibodies, FIG. 7 ). It is also possible to insert the sortase signal sequence within the loops of VSG (as done for FLAG peptide in Stavropoulos and Papavasiliou, 2010). However in such situations it is advisable to use the sortase donor sequence (e.g. LPXTGG) within the VSG loops and the sortase acceptor sequence (e.g. Ala-Ala) on the decorating small molecule, to increase specificity. Due to the dense coat of VSGs on the surface of trypanosomes, the small molecule is then densely displayed on the surface. Upon trypanosome injection into a mammalian host, the small molecule-conjugated VSG coat is exposed to the host immune system which mounts a similar strong and specific priming immune response against the exposed hapten (e.g. NP) as it does against the VSG in natural infection (FIG. 8 ). Boosting can then be achieved with hapten-VSG conjugate either on the full coat (FIG. 8 ) or formulations thereof (e.g. soluble haptenated VSG but other formulations as well) achieving a scalability that is unique to this vaccine platform.

Interestingly, primary responses elicited by different VSGs are not cross-reactive (i.e. antibodies raised to VSG2 do not cross-react with VSG3 etc. Pinger et al., Nat. Communications, 2017). This suggests that each specific VSG elicits a unique subset of B cell specificities (thus, a unique repertoire) which could be more or less potent toward a specific set of small-molecule haptens. In context of the invention, specific VSGs are selected for specific haptens, for the elicitation of optimal anti-hapten responses (with some VSGs better platforms for certain haptens—FIG. 7 ). For example, good anti-HA (pan-influenza) antibodies require engagement of IGHV1-69, a “rare” IGH within the general repertoire. A VSG that elicits IGHV1-69 might therefore be engaged preferably if one desired to use this method to elicit polyspecific anti-influenza antisera (e.g. in a “pan-flu” vaccine). Direct proof that VSGs elicit distinct B cell repertoires (and that this system can provide a range of different platforms depending on the preference of B cell to be elicited) is provided in FIG. 11 .

Biosafety concerns (e.g. disease causation) but also a need to block natural switching away from the haptenated VSG, dictate that derivatized trypanosome coats are inactivated (and thus unable to cause infection). The inventors have achieved this via UV-crosslinking of trypanosomes that lack the enzyme glycophosphatidylinositol phospholipase C (GPI-PLC) and are therefore dead—but with an intact VSG coat (trypanosomes wildtype for GPI-PLC disintegrate upon UV-inactivation as GPI-PLC cleaves the GPI linkage of each VSG off the membrane and sheds the coat (as depicted in FIG. 5 ). UV-crosslinking (of GPI-PLC-negative trypanosomes is achieved by pelleting cells from culture, washing with irradiation buffer (PBS supplemented with 55 mM Glucose), and resuspending in the same buffer to a density of 10⁷ cells/ml. 1 ml of this suspension is then aliquoted into each well of a 6-well tissue culture plate (Thermoscientific, 150239). Plates are UV-irradiated for 8 cycles, each cycle 30 S using a UVP crosslinker (Analytik Jena). Plates are swirled between irradiation cycles to ensure equal irradiation of trypanosomes. Irradiated cells are then resuspended at a concentration of 15×10⁶ cells/ml. 200 μl of this solution (3×10⁶ trypanosomes) can be injected intraperitoneally or sub-cutaneously into mice.

Overall, using this inactivation protocol and sortaggable VSGs, the inventors have generated an optimal and flexible platform for the immunogenic display of antigenic determinants toward the generation of antibodies to small-molecule haptens and peptides, which can be expanded to a wide variety of antigenic entities (e.g. lipids, nucleic acids, etc.) Proof of concept regarding generation of antibodies to small molecules (e.g. NP) is shown in FIG. 9 . Proof of principle that such antibodies can be raised against fentanyl and when generated protect against intoxication is provided in FIGS. 9 and 10 . A cartoon version of the overall method is illustrated for the small molecule 6-FAM in FIG. 6 .

An example of how to integrate the engineered VSGs of the invention into the T. brucei genome is provided in FIG. 3 .

Generalizability of the approach: For the purposes of this application, the inventors focus on active immunotherapy against fentanyl, an adulterant of synthetic heroin and the cause of the majority of drug overdoses in the United States. This is because the inventors have tools already available (fentanyl haptenated to LPXTGG so that it can be sortagged; anti-fentanyl antibodies to verify sortaggability—from M. Pravetoni). However it should be clear that this approach can easily be adapted to raise effective antibodies against other drugs and drug metabolites (e.g. acetaminophen metabolites which cause liver toxicity, small molecules that are the toxins causal to anaphylactic shock in certain foodstuff allergies etc.). The approach can also be used for the haptenation with peptides derived from pathogens (e.g. the NANP tandem repeat, a major antigenic determinant of the Circumsporozoite protein of Plasmodium falciparum) or with aberrantly-glycosylated peptides unique to cancer cells (e.g. mucin) which can be used as anti-cancer vaccines (PMID: 20403708).

Example 2: A Unique Prime-Boost Combination for the Generation of High-Quality Antibody Responses and B Cell Memory

The immunization scheme developed in the experiments of FIGS. 8 and 9 above was further tested. The present invention is based on a different way to prime and boost, that does not require adjuvant but appears to elicit cognate T-cell help to small molecules in a physiological fashion. Specifically, the inventors used a hapten-carrier combination in a prime-boost combination (e.g. fentanyl covalently attached to trypanosome VSG protein) but in two completely distinct biophysical entities: the entire VSG coat of a trypanosome (a micrometer size particle which is highly dense and repetitive) followed by the same carrier moiety but now in soluble form (FIG. 12 ).

It was found that the haptenated VSG particle, despite its size, presents a highly immunodominant surface that can elicit a highly specific antibody repertoire and a predominantly IgM response (FIG. 13A). Conversely, it was found that the same moiety but now used as the soluble form during the boost, can elicit high IgG titers (equivalent in proportion to the carrier as to the hapten and also memory (FIG. 13B). Neither the priming nor the boost step requires adjuvant, suggesting that the invention is based on an, to date, unknown physiological mechanism to generate high antibody titers and memory. Furthermore, the amounts used are about 250× lower than those used in standard protocols (e.g. 100 μg NP-CGG per mouse), and lead to titers that are long lasting (e.g. still increasing even one month after the first boost) (FIG. 13C).

Example 3: Specific Priming Boosting Protocol—Dosing and Timing

Herein it was generally dosed twice within 30 days as the priming step (day 0 and day 30). This dual step is not essential but may help activate rare B-cell receptors from the germline repertoire. Then the inventors boosted at day 60 or day 60 and day 90. The day 60 boost leads to a recall IgG response that continues to increase in titers for the subsequent 30 days (FIG. 13C).

As mentioned, a dose that is about 250×lower than that delivered with standard methods is sufficient in the protocol of the invention. However, dose escalation (increase) at the priming step is not necessarily beneficial and potentially even detrimental (injection of material representing more than 50 million cells results in decreased titers, compared to injection representing 3 or 10 million cells—FIG. 14 ). An optimized protocol includes a priming dose representing about 5 million cells per bout 20 g of body weight—it resulted in an IgG response that is substantial (about 15% of total IgG in a mouse (2.38 mg/ml total IgG in a mouse (Klein-Schneegans et al., 1989))— FIG. 15 ). Thus, a two-step prime/boost process should be used in higher organisms as well, starting with a small dose as stated above.

In the below protocol details on the successful immunization schedule, using fentanyl as an example, are provided.

Six 6-8 weeks old female C57BL/6J mice per group were immunized with 5E6 cells of UV-irradiated sortagged VSG3-fentanyl cells (VSG3-Fen) subcutaneously (s.c.) in 200 μl of PBS at days 0 and 30. As control, 6 mice were either left unimmunized or injected with non-tagged UV-irradiated VSG3 carrier.

Two boosts were done using non-adjuvanted soluble VSG3 or VSG3-Fen in PBS at days 60 and 90. Blood samples were collected from the facial vein 4 days before starting the immunizations (day −4), 2 days before the first (day 58) and 8 days after the first (day 68) and second boosts (day 88 and day 98) with soluble VSG3 or VSG3-Fen proteins. [This material can be prepared ahead of time, concentrated and frozen in PBS; it can be thawed and diluted in PBS at the proper dose, prior to injection].

Results are shown in FIG. 16 .

Specific IgG antibody titers against both Fen-hapten and VSG3 carrier protein in sera were measured by ELISA using 96-well plates coated with BSA-Fen conjugates or purified VSG3.

TABLE 1 Immunization Schedule: 1^(st) Immunization 2^(nd) Immunization 3^(rd) Immunization 4^(th) Immunization Group (Day 0, S.C.) (Day 30, S.C.) (Day 60, S.C.) (Day 90, S.C.) VSG3-Fen VSG3-Fen cells VSG3-Fen cells 100 μg VSG3-Fen in 100 μg VSG3-Fen in (5E6 cells, in PBS (5E6 cells, in PBS PBS (no adjuvant) PBS (no adjuvant) or PBS-glucose) or PBS-glucose) VSG3 (carrier VSG3 cells (5E6 VSG3 cells (5E6 100 μg VSG3 in 100 μg VSG3 in control) cells, in PBS) cells, in PBS) PBS (no adjuvant) PBS (no adjuvant) non-immunized

Example 4: Comparing Vesicles to UV Treated Coats

In order to compare long term efficacy of vaccination using vesicles to UV treated coats, immunization was carried out as described in Example 3 using vesicles containing VSG3-Fen to UV treated trypanosomal coats.

Anti-fentanyl IgG titers (normalized AUC values) were determined over a time course comprising day 2, 12, 22, 40 and 50 measurements; see FIG. 17 , priming on the left side was carried out with UV treated trypanosomal coats whereas vesicles were used on the right side. Boosting was carried out in both groups using soluble VSG as described before. A significantly stronger increase in IgG titers was found for vesicles.

Accordingly, vaccination using vesicles is apparently superior over vaccination using UV treated coats. Moreover, vesicles are more amenable to GMP production and have a longer shelf life. 

1. A method for eliciting an immune response, and/or for amplifying a pre-existing immune response, against an antigenic compound in a subject, comprising at least one priming-immunization step and at least one boosting-immunization step, wherein the at least one boosting-immunization step is performed after the at least one priming-immunization step, and wherein: (i) The at least one priming-immunization step comprises an immunization of the mammal with an array of immunization-carrier-fusions on a vesicle or bead, and wherein each immunization-carrier-fusion is composed of a carrier protein fused to the antigenic compound; and (ii) The at least one boosting-immunization step comprises an immunization of the mammal with the immunization-carrier-fusion used in (i) in a non-aggregated, preferably soluble form.
 2. The method of claim 1, wherein the immunization-carrier-fusion is an antigenic particle coated with an engineered variant surface glycoprotein (eVSG), wherein the eVSG comprises the antigenic compound covalently linked, optionally via a linker, to the N-terminus of the VSG carrier protein.
 3. The method of claim 1, wherein the immunization-carrier-fusion has the following (covalent) structure from N- to C-terminus: antigenic compound, a sortagging donor sequence, a sortagging acceptor sequence, a linker, a VSG protein sequence.
 4. The method of claim 1, wherein the antigenic compound is a disease associated antigen, for example a cancer antigen, an allergen or a viral immunogen, or is a dependency causing substance selected from (i) delta tetrahydrocannabinol (THC) or Synthetic cannabinoids, such as classical cannabinoids, non-classical cannabinoids, hybrid cannabinoids, aminoalkylindoles, and eicosanoids; for example Δ9-THC HU-210, (C8) CP 47,497, JWH-018, AM-2201 (Fluorinated JWH-018), UR-144, XLR-11 (Fluorinated UR-144), APICA, STS-135 (Fluorinated APICA). AB-PINACA, PB-22, 5F-PB-22 (Fluorinated PB-22); or (ii) methamphetamine and derivatives thereof such as 3,4-methylenedioxy-methamphetamine (MDMA)Ecstasy/Molly; or (iii) a synthetic cathinone like alpha-pyrrolidinopentiophenone (alpha-PVP); or (iv) an opioid including heroin, synthetic opioids such as fentanyl or related compounds such as carfentanyl, and other opioid pain relievers, such as oxycodone (OxyContin®), hydrocodone (Vicodin®), codeine, morphine, desomorphine (Krokodil); or (v) steroids (anabolic substances), or is nicotine.
 5. The method of claim 1, to wherein the carrier protein is a VSG protein derived from Trypanosoma brucei.
 6. The method of claim 1, wherein the vesicle or bead is coated with more than one immunization-particle fusions, preferably more than 10 immunization-particle fusions per particle.
 7. The method of claim 1, wherein the immunization-carrier used in the method is a VSG protein, and wherein the VSG protein used as carrier in (i) and (ii) are derived from the same VSG protein type, for example the VSG in both (i) and (ii) are VSG3 of Trypanosoma brucei.
 8. The method of claim 1, wherein the method comprises at least two priming-immunization steps, and wherein all of the priming immunization steps are performed prior to any of the boosting-immunization steps.
 9. The method of claim 1, wherein the method comprises at least two boosting-immunization steps, and wherein all of the boosting immunization steps are performed after any of the priming-immunization steps.
 10. The method of claim 1, wherein the subject is selected from an animal having an adaptive immune system, such as vertebrates such as birds (chicken), and mammals such as mouse, rabbit, camel, goat, rat, dog, cat, monkey, hamster or other mammals, or is a human, such as a human patient in need of an elicited or amplified immune response.
 11. A non-therapeutic method for generating antibodies against an antigenic-compound, the method comprising performing in a non-human animal the method of claim 1, and isolating from the non-human animal antibodies or B-cells producing antibodies against the antigenic compound.
 12. An antibody, or an antibody producing B-cell, generated and/or isolated according to a method of claim
 11. 13. An immunization kit, the immunization kit comprising (a) an aggregate of immunization-carrier protein on a vesicle or bead as recited in claim 1, and (b) a non-aggregated, preferably soluble form, of the immunization-carrier protein of (a); wherein the immunization-carrier protein of (a) and (b) are capable to be fused to an antigenic compound to obtain an immunization-carrier-fusion recited in claim
 1. 14. A pharmaceutical composition, comprising the immunization-carrier-fusion claim
 1. 